inauguraldissertation - FreiDok plus

Structural and functional characterization of

a novel Ammonium Transport protein from

“Candidatus Kuenenia stuttgartiensis”

INAUGURALDISSERTATION

zur Erlangung des Doktorgrades

der Fakultät für Chemie, Pharmazie und Geowissenschaften

der Albert-Ludwig-Universität Freiburg im Breisgau

vorgelegt von

Camila José Hernández Frederick

aus Caracas, Venezuela

Freiburg 2011

Vorsitzender des Promotionsausschusses: Prof. Dr. Rolf Schubert

Referent: Dr. Susana Andrade

Korreferent: Prof. Dr. Oliver Einsle

Datum der Promotion: 23.09.2011

For my mother, my father and my little brother Los quiero con todo mi corazón

Table of contents

Camila Hernández

4

1 Zusammenfassung ............................................................................................................. 7

2 Summary ............................................................................................................................... 8

3 Introduction ......................................................................................................................... 9

3.1 Nitrogen cycle and biological relevance ................................................................. 9

3.2 Anaerobic ammonium oxidation (anammox) ................................................... 11

3.2.1 Anammox bacteria ................................................................................................................ 14

3.3 Ammonium transport proteins (Amt) ................................................................. 16

3.3.1 Amt protein structures ........................................................................................................ 17

3.3.2 Ammonia/ammonium transport mechanism ............................................................ 21

3.3.3 Regulation of Amt proteins ................................................................................................ 26

3.3.4 Multiplicity of Amt proteins .............................................................................................. 29

3.3.5 The Amt protein Ks-Amt5 from “Ca. Kuenenia stuttgartiensis” .......................... 30

3.4 Histidine kinases ......................................................................................................... 31

3.4.1 Characteristic sequence motifs and function .............................................................. 33

3.4.2 Classification of histidine kinase proteins ................................................................... 36

3.4.3 Structure of the cytoplasmatic portion of the sensor histidine-kinase TM083 from Thermotoga maritima ............................................................................................................. 38

3.5 Aims of this work ......................................................................................................... 40

4 Materials and Methods .................................................................................................. 42

4.1 Materials ......................................................................................................................... 42

4.1.1 Chemicals .................................................................................................................................. 42

4.1.2 Detergents ................................................................................................................................ 42

4.1.3 DNA and Protein Weight Markers ................................................................................... 42

4.1.4 Enzymes .................................................................................................................................... 42

4.1.5 Bacterial strains ..................................................................................................................... 43

4.1.6 DNA oligonucleotides ........................................................................................................... 44

4.1.7 Plasmids: The pET vector system ................................................................................... 44

4.2 Methods ........................................................................................................................... 47

4.2.1 Molecular biology .................................................................................................................. 47

4.2.1.1 Polymerase Chain Reaction (PCR)............................................................................... 47

4.2.1.2 Site-directed mutagenesis .............................................................................................. 49

4.2.1.3 DNA digestion with restriction endonucleases ...................................................... 49

4.2.1.4 DNA ligation ......................................................................................................................... 50

4.2.1.5 Agarose gel electrophoresis ........................................................................................... 50

4.2.1.6 Extraction of DNA from agarose gels ......................................................................... 51

4.2.1.7 DNA Sequence Analysis ................................................................................................... 52

Table of contents

Camila Hernández

5

4.2.2 Microbiological methods .................................................................................................... 52

4.2.2.1 Escherichia coli cultivation ............................................................................................ 52

4.2.2.2 Production and transformation of E. coli competent cells ................................. 52

4.2.2.3 Plasmid preparation ......................................................................................................... 53

4.2.2.4 Protein production in E. coli .......................................................................................... 54

4.2.3 Protein biochemistry ............................................................................................................ 54

4.2.3.1 Cell disruption and preparation of purification samples ................................... 54

4.2.3.2 Solubilization of membranes ......................................................................................... 55

4.2.3.3 Affinity chromatography ................................................................................................. 57

4.2.3.4 Size exclusion chromatography (SEC) ....................................................................... 58

4.2.3.5 Protein concentration determination ........................................................................ 60

4.2.3.6 SDS PAGE electrophoresis .............................................................................................. 61

4.2.3.7 Coomasie Brilliant Blue (CBB) staining ..................................................................... 63

4.2.3.8 Phosphorylation assay ..................................................................................................... 64

4.2.3.9 Western blot......................................................................................................................... 66

4.2.3.10 Blue Native PAGE (BN-PAGE) ..................................................................................... 68

4.2.3.11 Isothermal titration calorimetry ............................................................................... 69

4.2.3.11.1 ITC experiments with Ks-Kin................................................................................... 72

4.3 Protein crystallography ............................................................................................ 73

4.3.1 Crystallization ......................................................................................................................... 73

4.3.2 Crystallization of Ks-Amt5.................................................................................................. 74

4.3.3 Finescreens .............................................................................................................................. 75

4.3.4 Structure determination by X-ray crystallography .................................................. 76

4.3.5 Crystal arrangement ............................................................................................................. 76

4.3.6 X-ray diffraction by protein crystals .............................................................................. 77

4.3.7 The electron density function ........................................................................................... 80

4.3.8 Molecular replacement ........................................................................................................ 82

4.3.9 Structure determination of Ks-Amt5.............................................................................. 85

4.3.9.1 Cryo-cooling ......................................................................................................................... 85

4.3.9.2 Data collection and processing ..................................................................................... 86

4.3.9.3 Structure solution .............................................................................................................. 86

4.3.9.4 Model building and refinement .................................................................................... 87

4.4 Graphical representations ....................................................................................... 87

5 Results and discussion .................................................................................................. 88

5.1 Sequence analysis of Ks-Amt5 ................................................................................. 88

5.2 Cloning and mutagenesis of Ks-Amt5 ................................................................... 91

5.3 Protein production ..................................................................................................... 92

5.4 Protein purification .................................................................................................... 94

5.4.1 Ks-Amt5 ..................................................................................................................................... 94

5.4.2 Ks-Kin and variants ............................................................................................................... 97

5.5 Crystallization of Ks-Amt5 ...................................................................................... 100

Table of contents

Camila Hernández

6

5.6 Crystallization of Ks-Kin .......................................................................................... 102

5.7 Data collection and processing ............................................................................. 103

5.8 Overall structure and crystal packing ................................................................ 104

5.9 Ks-Amt5 monomer .................................................................................................... 107

5.10 Structural comparison of Ks-Amt5 with other Amt proteins .................. 111

5.11 Small Angle X-ray Scattering ............................................................................... 115

5.12 Functional studies .................................................................................................. 116

5.12.1 Thermodynamic characterization of Ks-Kin ........................................................... 116

5.12.2 Phosphorylation analysis of kinase activity of Ks-Amt5 .................................... 118

5.13 Remarks on the possible mechanism of transport for Ks-Amt5 ............ 122

5.14 Future perspectives ............................................................................................... 124

6 Appendix .......................................................................................................................... 126

6.1 Abbreviations ............................................................................................................. 126

6.2 Units ............................................................................................................................... 127

6.3 Prefixes ......................................................................................................................... 128

6.4 Amino acids ................................................................................................................. 128

6.5 Ks-Amt5 DNA sequence ........................................................................................... 129

6.6 Ks-Amt5 amino acid sequence .............................................................................. 130

7 References ....................................................................................................................... 131

8 Acknowledgements – Danksagung – Agradecimientos ...................................... 149

9 Curriculum Vitae ............................................................................................................ 152

Zusammenfassung

Camila Hernández

7

1 Zusammenfassung

Die Assimilierung von Stickstoff ist ein essenzieller biologischer Prozess.

Weitverbreitete Amt-Proteine katalysieren die Aufnahme von reduzierten Stickstoff

in Form von Ammonium. Sie sind in der Lage, Ammonium über zelluläre

Membranen zu transportieren und machen den reduzierten Stickstoff damit direkt

zugänglich für die Synthese von Biomolekülen. Trotzdem schon hochauflösende

Kristallstrukturen existieren, bleibt die Art des Substrats Gegenstand kontroverser

Diskussionen. Das Anammox-Bakterium “Candidatus Kuenenia stuttgartiensis“,

welches unter anaeroben Bedingungen in der Lage ist, Ammonium zu Stickstoff zu

oxidieren, besitzt fünf Kopien von amt-Genen in seinem Genom. Eine dieser Kopien

kodiert für ein untypisches, bisher unbeschriebenes Amt-Protein (Ks-Amt5). Neben

den typischen Charakteristica eines Ammoniumtransport-Proteins besitzt es eine

lösliche Domäne, welche als Histidin-Kinase identifiziert werden konnte. Histidin-

Kinasen sind Bestandteil eines Zweikomponentensystems zur Signalübertragung.

Sie sind in der Lage, extrazelluläre Signale zu erkennen, was zu alternierender

Aktivität von Autokinase und Autophosphatase führt. In dieser Arbeit wurde die

Kristallstruktur des Proteins Ks-Amt5 mit einer Auflösung von 2.1 Å gelöst. Das

Protein weist Homologien zu anderen Ammoniumtransport-Proteinen von

Escherichia coli (AmtB) oder Archaeoglobus fulgidus (Amt1) auf. Zusätzlich wurden

funktionelle Studien durchgeführt, welche die Kinase-Aktivität in Abhängigkeit der

Ammonium-Konzentration beschreiben. Mit diesen Ergebnissen kann ein möglicher

Reaktionsmechanismus für dieses spezielle Amt-Protein vorgeschlagen werden.

Summary

Camila Hernández

8

2 Summary

Nitrogen assimilation is an essential biological process. The ubiquitous Amt proteins

are involved in the uptake of reduced nitrogen in the form of ammonium. The Amt

proteins are able to transport ammonium across cellular membranes thus making

this reduced form of nitrogen directly accessible to organisms for assimilation.

Although, high resolution crystal structures are available the nature of the substrate

being transported is still on debate and controversially discussed. The anammox

bacteria “Candidatus Kuenenia stuttgartiensis” which is able to oxidize ammonium

under anoxic conditions to produce dinitrogen gas posseses five copies of amt genes

in the genome. One of these genes encodes for an exceptional and undescribed Amt

protein (Ks-Amt5). This protein presents besides the characteristic features of

ammonium transport proteins an extramembrane domain identified as a histidine

kinase protein. Histidine kinases are one of the basic components of two-component

signal transduction system. These proteins can recognize external signals which

lead to an alteration of its autokinase and autophosphatase activity. In this work,

Ks-Amt5 is structurally and functionally studied. By means of X-ray crystallography

the Ks-Amt5 structure was determined at 2.1 Å resolution. Ks-Amt5 presents

conserved topological and structural characteristics to its counterparts in

Escherichia coli (AmtB) and Archaeoglobus fulgidus (Amt1). In addition, functional

studies revealed that the kinase activity is linked to the variations in ammonium

concentrations. With this finding a possible mechanism for this remarkable protein

is proposed.

Introduction

Camila Hernández

9

3 Introduction

3.1 Nitrogen cycle and biological relevance

Nitrogen is an essential element in nature. It is the most frequent element in Earth’s

atmosphere, constituting 79% of air in the form of dinitrogen (N2) (Jetten et al.,

2009). Nitrogen is also important for living organisms, being found as a bound

component of nucleic acids, amino acids and other biomolecules, such as amino-

saccharides (Falkowski et al., 1998). Although nitrogen is highly abundant, its

bioavailability is very low due to the fact that most organisms, including plants and

animals, cannot metabolize atmospheric dinitrogen. Its characteristic triple bond

makes the inert gas dinitrogen the most stable form of nitrogen; therefore, its

conversion to further reduced states requires high amounts of energy (bond

dissociation energy 946 kJ mol-1) (Rees et al., 2005).

However, some microorganisms such as the diazotrophic organisms, are capable of

reducing dinitrogen (N2) into more accessible forms, such as ammonia (NH3) and

ammonium (NH4+) (Figure 1). This process known as biological nitrogen fixation is

of great importance to the environment and it is catalyzed by a broad class of

enzymes called nitrogenases (Rees & Howard, 2000; Dixon & Kahn, 2004; Rees et al.,

2005). Fixed and reduced nitrogen in the form of NH3/NH4+ can then be directly

assimilated for biosynthesis of biomolecules and incorporated as biomass.

The nitrification process describes the oxidation of NH3/NH4+ to nitrite (NO2

-) by

ammonia-oxidizing bacteria (AOB), such as Nitrosomonas, or further to nitrate

(NO3-) by nitrite-oxidizing bacteria (NOB), such as Nitrobacter. Nitrification is

carried out under strict aerobic conditions (Schmidt et al., 2001) or anaerobically by

selected species given an external supply of NO2- (N2O4) (Arp et al., 2007). This

process is catalyzed by three enzymes, the ammonia oxygenase, the hydroxylamine

oxidoreductase and the nitrite oxidase (Klotz & Stein, 2008).

Introduction

Camila Hernández

10

Figure 1: Basic steps of the Nitrogen Cycle. Nitrogen fixation: dinitrogen is

reduced to bio-accessible forms (ammonia/ammonium) by microorganisms called

diazotrophs. Nitrification: ammonia and ammonium are oxidized to nitrite by

ammonium-oxidizing bacteria (AOB) and to nitrate by nitrite-oxidizing bacteria

(NOB). The products of both processes, nitrogen fixation and nitrification, can be

then assimilated by other microorganisms and plants. The denitrification and

anammox processes close the cycle, converting the reduced and oxidized forms of

nitrogen back to gaseous dinitrogen.

The nitrogen cycle (Figure 1) is completed by the anaerobic process of

denitrification. During denitrification, nitrate and nitrite are reduced back to

gaseous dinitrogen. The process comprises four steps: (1) Nitrate is reduced to

Introduction

Camila Hernández

11

nitrite by the enzyme nitrate reductase, (2) nitrite is reduced further to nitric oxide

(NO) by the nitrite reductase, (3) nitric oxide is reduced to nitrous oxide (N2O) by

the nitric oxide reductase, and (4) the enzyme nitrous oxide reductase carries out

the last step of reduction of N2O to dinitrogen (Zumft et al., 1997; Einsle & Kroneck,

2004).

Recently, a fourth process was found to contribute to the production of N2 (Jetten et

al., 2005a). This process called anaerobic ammonia oxidation (anammox) is an

alternative route in the nitrogen cycle and it is found among one group of bacteria

known as Planctomycetes.

3.2 Anaerobic ammonium oxidation (anammox)

The anammox reaction is a microbiological process in which ammonium is oxidized

to dinitrogen gas coupled with the reduction of nitrite under strict anaerobic

conditions (Arp et al., 2007; Jetten et al., 2005b; Klotz & Stein, 2008). Important

intermediates of this reaction are hydrazine (N2H4), a toxic and high-energetic

compound, and hydroxylamine (NH2OH), a compound also used as solid propellant

(Jetten et al., 2002).

NH4++1.32 NO2

-+0.066 HCO3+0.13 H+ 0.26 NO3-+1.02 N2+0.066 CH2O0.5N0.15 +2.03 H2O

Scheme 1: Overall reaction of the anammox process and its stoicheometry (Strous et al., 1998).

While ammonium, nitrite and nitrate are primarily nitrogen sources to sustain

metabolic reactions, in higher concentrations they also contribute to the

eutrophication of water environments (Ye & Thomas, 2001). The anammox process

is considered as an important mechanism that removes undesired ammonium from

municipal and industrial waste water (Jetten et al., 2005b; Kuenen, 2008). Recently,

it has been estimated that 50% of the fixed nitrogen removal from the ocean is due

to the anammox (Strous et al., 2006).

Introduction

Camila Hernández

12

In 2008, van Niftrik et al., proposed a biochemical model (Figure 2) for the

anammox reaction. In this model, nitrite is reduced to nitric oxide by a cytochrome

c- and cytochrome d1-containing nitrite reductase (NirS). Further, nitric oxide and

ammonium are presumed to be combined into hydrazine by the hydrazine

hydrolase (HH). Finally, the hydrazine is oxidized to N2 by the

hydrazine/hydroxylamine oxidoreductase (HAO/HZO), an octaheme cytochrome c

enzyme. This oxidation step produces the release of four electrons, which are

transferred first to soluble cytochrome c electron carries and later to ubiquinone,

cytochrome bc1 complex (complex III) and other soluble cytochrome c electron

carries and finally back to nitrite reductase and hydrazine hydrolase. Consequently,

this process generates a proton motive force that could be used for the production

of energy by means of ATP synthesis.

Figure 2: Schematic representation of the ultrastructure of an anammox bacteria and

proposed biological model of the anaerobic ammonium oxidation process. A. Morphology of

anammox bacteria showing the different subcellular compartments and membranes. B. Postulated

coupling of the anammox reaction to the anammoxosome membrane. Nir: nitrite reductase

(cytochrome cd1); hh: hydrazine hydrolase; hao: hydrazine/hydroxylamine oxidoreductase

(octaheme cytochrome c); cyt: mono- or diheme cytochrome c electron carries; bc1: cytochrome bc1

complex (complex III); Q: coenzyme Q (ubiquinone). Result of this reaction is the production of

dinitrogen with an increasing proton motive force and the consequent synthesis of ATP by ATPases.

Reprinted from van Niftrik et al., 2008.

Introduction

Camila Hernández

13

The anammox reaction takes place in the anammoxosome (Figure 2A), an

intracytoplasmatic compartment that comprises 50-70% of the total cell volume.

The anammoxosome is surrounded by a dense membrane that contains unique rigid

lipids. These structurally unusual lipids are called ladderanes (Figure 3) and are

formed by the fusion of cyclobutane and cyclohexane rings (van Niftrik et al., 2004).

It is supposed that the ladderane lipids contribute to the limited diffusion of the

anammoxosome membrane thus preserving the concentration gradients during

replication and protecting the rest of the cell against toxic anammox intermediates

(Sinninghe-Damsté, 2002). Further, it has been found that the biosynthesis of these

ladderanes is exclusive to anammox bacteria. Therefore, they are currently used as

biomarkers for the presence of these organisms in environmental samples (Kuypers

et al., 2003).

Figure 3: Structure and composition of the ladderane lipids from anammox bacteria. Reprinted

from Jetten et al., 2009.

Introduction

Camila Hernández

14

3.2.1 Anammox bacteria

Anammox bacteria are chemolitoautothrophic organisms that use bicarbonate as a

sole carbon source for the biosynthesis of cell biomass and derive their energy from

the conversion of ammonium and nitrite into dinitrogen (van Niftrik et al., 2004). As

members of the Planctomycetales order from the bacterial domain they are

considered an ecologically and environmentally important group of microorganisms

(Jetten et al., 2009).

Anammox bacteria were first discovered in the 1990’s in the Gist-Brocades

fermentation plant, Netherlands (Kuenen & Jetten, 2001). From that time on,

anammox bacteria have been found in many different environments, such as coastal

sediments, lakes, marine suboxic zones and wastewater treatment plants (Schmid et

al., 2007). All anammox organisms belong to the monophyletic group called

Brocadiales. So far, only five genera of anammox bacteria with the status

“Candidatus” have been described: “Ca. Brocadia” (Strous et al. 1999; Kuenen &

Jetten, 2001; Kartal et al., 2008), “Ca. Kuenenia” (Schmid et al., 2000; Strous et al.,

2006), “Ca. Anammoxoglobus” (Kartal et al., 2007), “Ca. Jettenia” (Quan et al., 2008)

and “Ca. Scalidua” (Kuypers et al., 2003; Schmid et al., 2003; van de Vossenberg et

al., 2008).

Anammox bacteria are coccoid shaped bacteria (Figure 4) with a diameter of 800

nm and are characteristically slow growers with a variable doubling time from 10-

20 days (Jetten et al., 2009). Additionally, it is known that concentrations above 2

µM oxygen can inhibit their metabolism, as a consequence they are also classified as

obligate anaerobes (van Niftrik et al., 2004).

Introduction

Camila Hernández

15

Figure 4: Electron microscopy representation of a “Candidatus Kuenenia

stuttgartiensis” cell. The white dots show the distinct subcellular

compartments, including the anammoxosome, where the anammox

reaction takes place. The scale bar represents 200 nm. Reprinted from

Kuenen, 2008.

“Ca. Kuenenia stuttgartiensis” is the model organism for this study. In 2006, Strous

et al., published a nearly complete genome of “Ca. Kuenenia stuttgartiensis”. This

constituted the first sequenced genome of an anammox bacterium. The 4.2

megabase genome was used to decipher the biochemical pathway of anaerobic

ammonium oxidation. In this genome, 200 genes were detected to be relevant for

respiration and anammox catabolism (Strous et al., 2006). Additionally, five amt

genes were found to codify for ammonium transport proteins.

Introduction

Camila Hernández

16

3.3 Ammonium transport proteins (Amt)

Ammonium (NH4+/NH3) is a product of nitrogen fixation and a direct nitrogen

source for many organisms, such as bacteria, fungi and plants. It is used as a

substrate in metabolic reactions that involve the enzymes glutamine synthetase

(GS), glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH), resulting

in the biosynthesis of the amino acid glutamine. From glutamine, other amino acids

can be synthesized upon transamination reactions (Purich, 1998). However, in high

concentrations, ammonia can be cytotoxic to animals.

Due to this crucial metabolic role, the transport of NH4+/NH3 is an essential

biological process in microorganisms and plants (Broach et al., 1976; van Dommelen

et al., 2001). In mammals, NH4+/NH3 transport is also essential to kidney physiology

for the maintenance of pH and in renal ammonia secretion (Knepper, 1991).

Ammonia is a hydrophobic gas that can diffuse freely across biological membranes

(Lande et al., 1995). However, in aqueous solution, NH3 is in equilibrium with the

protonated form NH4+ controlled by a pKa=9.25. Thus, at physiological pH of about

7.5, ammonia exists mainly as the membrane impermeable cation NH4+. A dedicated

transport protein is then necessary for the accessibility of NH4+ to convey metabolic

needs.

Proteins involved in the transport of ammonia/ammonium across cellular

membranes belong to the Amt/Rh family. This family of integral membrane proteins

is composed by the Ammonium transport proteins (Amt), found in bacteria, archaea

and plants, and their homologues Rhesus proteins (Rh) found in animals.

Amt proteins consist of 400-600 amino acids in length with a conserved core of 10-

12 transmembrane helices (Marini et al., 1994; Ninnemann et al., 1994; Thomas et

al., 2000a) and are mainly expressed at low substrate (ammonia/ammonium)

concentrations (Kleiner, 1985a). Several functions have been associated to these

proteins, being the high-affinity transport of NH4+ across the membrane the most

relevant. Additionally, it has been found that Amt proteins are required for optimal

Introduction

Camila Hernández

17

growth of some microorganisms at low pH (Marini et al., 1997; Soupene et al.,

1998). Recently, apart from transport function, Amt proteins have been found to act

as ammonium sensors in the regulation of nitrogen metabolism (Javelle et al., 2004;

Javelle & Merrick, 2005).

3.3.1 Amt protein structures

Despite intense research efforts on Amt proteins, so far only four crystal structures

have been solved. In 2004, Khademi et al., and in parallel Zeng et al., published the

first crystal structure of an Amt protein. The 1.4 Å resolution structure of AmtB from

E. coli confirmed the predicted trimeric stoichometry of these proteins (Blakey,

2002), and gave initial insights on how transport could work. Further, in 2005,

Andrade et al. published the Af-Amt1 structure, one of three Amt proteins from the

hyperthermophilic archaeon Archaeoglobus fulgidus, at a resolution of 1.54 Å. More

recently, two crystal structures of Rh proteins were solved at high resolution, the

Ne-Rh50 protein from Nitrosomonas europaea (Li et al., 2007, Lupo et al., 2007) at

1.3 Å resolution and the human RhCG (Gruswitz et al., 2010) at 2.1 Å resolution. All

these structures share a high degree of sequence and structural homology with

various conserved amino acids supposed to be involved in the ammonium transport.

Amt proteins are highly stable homotrimers containing 11-12 hydrophobic

transmembrane -helices per monomer. The sequence of Ec-AmtB presents a

twelfth N-terminal transmembrane helix as part of a leader peptide (residues M1-

A22) that is removed upon maturation and insertion of the protein into the cell

membrane (Khademi et al., 2004). However, the structure of human RhCG protein

presented an additional N-terminal transmembrane helix located at the interface of

each subunit. This additional N-terminal helix is conserved among higher

eukaryotes (Gruswitz et al., 2010).

Both crystal structures, Ec-AmtB (Figures 5A and 5C) and Af-Amt1 (Figures 5B and

5D), show a pseudo-twofold symmetry with a pseudo-twofold axis in the plane of

the membrane, formed by helices TM1-TM5 (counted from the N-terminus) and

Introduction

Camila Hernández

18

TM6-TM10, which is conserved among all Amt proteins (Khademi et al., 2004; Zeng

et al., 2004; Andrade et al., 2005). Additionally, they present an N-out/C-in topology

that follows the positive-inside rule for membrane proteins (von Heijne & Gavel,

1988), where the N-terminus is exposed to the periplasm and the C-terminus is

exposed to the cytoplasm. The final C-terminal helix, TM11, is tilted with respect to

the membrane plane and surrounds the monomer in the outer surface, holding

together the two pseudo-symmetric halves formed by helices TM1-TM5 and TM6-

TM10.

Figure 5: Structure of the Amt monomer from E. coli and A. fulgidus. A. Ec-AmtB monomer

(PDB accession code: 1U7G) and B. Af-Amt1 (PDB accession code: 2B2H) showing eleven

transmembrane -helices (TM1-TM11) in cartoon representation. The protein chain is colored

from blue at the N-terminus to red at the C-terminus. The cellular membrane is represented by

grey lines. C and D show the pseudo two-fold symmetry of the monomers of AmtB and Amt1,

Introduction

Camila Hernández

19

respectively.

The threefold symmetry of the homotrimeric protein (Figure 6) is given by the

interaction of residues from helices TM1, TM6, TM7, TM8 and TM9 of one monomer,

with residues of neighboring monomers from helices TM1, TM2 and TM3. Further,

the interfaces between monomers are highly hydrophobic. This fact led Khademi et

al. (2004) to suggest that the monomer is stable in the membrane during synthesis

before trimer formation.

Figure 6: Structure of the Af-Amt1 trimer. A. Side view of the molecular surface of the Amt1

trimer, the monomers are colored in silver, light and dark blue. B. View of the Amt1 trimer from the

extracellular side, the transmembrane helices that are involved in the trimer formation are labeled in

one of the monomer-monomer interaction surfaces. PDB accession code: 2B2H.

Every monomer presents two vestibules formed by helices TM1-TM10 on its

extracellular and intracellular side (Figure 7A). The putative substrate recruitment

site in each monomer, resides on the extracellular vestibule of the trimer. The Ec-

AmtB extracellular vestibule shows several carbonyl oxygens that form a funnel for

substrates (Khademi et al., 2004). In the inner part of this vestibule, two highly

conserved residues, W137 and S208 (numbered according to the Af-Amt1

sequence), are believed to be involved in the binding of NH4+. It is thought that NH4

+

could be selectively recruited at this position by the formation of a cation-π

Introduction

Camila Hernández

20

interaction with W137 in addition to a hydrogen bond to S208 (Andrade et al.,

2005). Below this NH4+ binding site, the side chains of two conserved residues, F96

and F204, constrict the channel to the cytoplasmatic side, indicating a possible

structural rearrangement upon substrate translocation (Figure 7B). Additionally,

the hydrophobic nature of the protein lumen leading to the cytoplasm could be

verified through pressurization experiments with the inert gas xenon (Andrade et

al., 2005). The one exception in this hydrophobic lumen is the presence of two

conserved histidine residues, H157 and H305, the imidazole rings of which are

arranged in an unusual manner forming a lateral hydrogen bond between their -

nitrogen atoms. In 2006, Javelle et al., reported the importance of this His pair in

substrate conductance.

Figure 7: Inside view of an Af-Amt1 monomer. A. The surface of the monomer is represented in

blue, where two vestibules are visible (shown by arrows), one extracellular and one intracellular

towards the cytoplasm. The cell membrene is represented by grey lines. B. Detail of A. The putative

recruitment site is at Trp137 and Ser208. Substrate passage is blocked by the “phenylalanine gate”

at Phe96 and Phe204. Following is the hydrophobic channel surrounded by hydrophobic residues

Introduction

Camila Hernández

21

except for the conserved coplanar His157 and His305. PDB accession code: 2B2H.

Regardless of the high structural similarities between Ec-AmtB and Af-Amt1,

especially in the transmembrane regions, significant differences were found in the

intracellular and extracellular loop regions. Contrary to Ec-AmtB, in the Af-Amt1

structure the entire protein and, for the first time, the C-terminal region was visible

and ordered in the crystal structure (Andrade et al., 2005). This C-terminal region

was later shown in Amt1;1 from Arabidopsis thaliana to be functionally important,

having an allosteric regulatory function for the transport activity (Loqué et al., 2007;

Loqué et al., 2009). The allosteric regulation is presumed to be controlled by the

phosphorylation of a conserved tyrosine residue located at the C-terminal region

( u hse, 2004; Loque et al., 2007). The phosphorylation event triggers a switch in

the ammonium transport protein, from an active state to an inactive state. This

change between active to inactive states, keeps the cell from incorporating too much

ammonium that in too high concentrations becomes toxic to the cell (Hess et al.,

2006; Szczerba, 2008).

3.3.2 Ammonia/ammonium transport mechanism

The knowledge gained from the high-resolution crystal structures of some Amt

family members represented a potential significant progress in the study of this

family of proteins. However, the understanding of the transport mechanism as well

as the identity of the substrate being transported (NH3 versus NH4+) remain

controversial and until now are not completely understood. So far, several models

for the transport mechanism by Amt proteins have been described. However, until

now, none of them have entirely explained the results obtained by the different

experiments.

In 1985, Kleiner, proposed a secondary active transport with co-transport of NH3/

H+. This system would be electrogenic, driven by an electrochemical gradient and

dependent on the proton-motive force and on the membrane potential. This

assumption was supported by uptake measurements of the 14C-labelled substrate

Introduction

Camila Hernández

22

analogue, methylamine (MA) in Amt proteins from yeast (Marini et al., 1994; Marini

et al., 1997) and Arabidopsis thaliana (Ninnemann et al., 1994; Gazzarrini et al.,

1999) where accumulation of intracellular MA was observed. Further, electrogenic

transport was proved in an oocyte system by voltage-clamp experiments with the

protein LeAMT1;1 from Lycopersicon esculentum (Ludewig et al., 2002) and the

RhBG glycoprotein (Nakhoul et al., 2005). These experiments demonstrated active

uptake of NH4+, showing a voltage-dependent current induced by the increase in

ammonium uptake upon increase of the external concentration of ammonium.

However, through these experiments it was not possible to discriminate the nature

of the transport between symport (NH3/H+) and uniport (NH4+). Therefore, this

model is still discussed and remains so far unproven.

Another model was proposed by the group of Sidney Kustu, which suggests that Amt

proteins work as gas channels facilitating the diffusion of the uncharged specie NH3.

This model was assumed after the results of in vivo studies made with whole cells of

E. coli (Soupene et al., 1998) and Salmonella typhimurium (Soupene et al., 2002),

where no accumulation of MA in the cytoplasm was observed, thus suggesting

diffusion of NH3 across the membrane. Moreover, first functional studies made with

Ec-AmtB proteins reconstituted into proteoliposomes (Khademi et al., 2004) in

combination with the independent observation of the hydrophobic nature of the

channel revealed by the crystal structures of Ec-AmtB and Af-Amt1 (Khademi et al.,

2004; Zeng et al., 2004; Andrade et al., 2005) supported the view of these proteins

as gas channels.

In these functional studies, Khademi et al. (2004), used a fluorescent pH-sensitive

dye, 5-carboxyfluorescein (CF) inside the proteoliposomes; it was then observed

that the internal pH of the proteoliposomes increased upon uptake of the substrate,

leading to the conclusion that NH3 is transported and becomes protonated to NH4+

(pKa=9.25) inside the proteoliposome causing the observed increase in pH. Based

on these findings and the fact that the select/recruitment site shows to bind NH4+

and not NH3, it was hypothesized that NH4+ would have to be deprotonated on the

Introduction

Camila Hernández

23

extracellular side and then reprotonated again in the cytoplasm, being translocated

as NH3 (Figure 8A) (Khademi & Stroud, 2006). However, this result represents a net

antiport of NH4+ versus H+ against a proton gradient, which encounters an energetic

problem (Andrade & Einsle, 2007). Due to the reverse H+ flow, the proton motive

force decreases and therefore energy would be required in the form of ATP to

perform such a type of transport. Consequent experimental data questioned the

model of Amt proteins as gas channels. In 2007, Fong et al., from the group of Kustu,

inferred uptake of NH4+ in a variant of Ec-AmtBW148L using a washed cell transport

assay with 14C-labelled MA. By means of these experiments the gas channel model

was questioned.

Figure 8: Critical view on the gas transport mechanism for Amt proteins.

A. At physiological pH values ammonium is mainly present as NH4+. The

uniport transport of NH3 requires the deprotonation of NH4+ in the periplasm

(extracellular) and reprotonation in the cytoplasm (intracellular). Thus, this

mechanism results in a net antiport of NH4+ versus H

+ that has no

physiological relevance. B. An alternative model showing a net uniport of

NH4+ occurring necessarily as a symport of NH3 and H

+. Adapted from

Andrade & Einsle, 2007.

Introduction

Camila Hernández

24

As stated, all transport mechanisms mentioned contradict themselves based on the

different experimental data. In addition, some of these functional studies,

particularly those presented by Khademi et al. (2004), were not reproducible

(Javelle et al., 2007), leading to a continuous debate about the ammonia vs.

ammonium transport.

A variation model was proposed to explain both observed hypotheses of

electrogenic transport and passive diffusion of NH3. This globalizing mechanism

includes the widely accepted view of deprotonation of NH4+ before entering the

hydrophobic pore and reprotonation in the cytoplasm after translocation of the

substrate. The model suggests that the substrate translocation occurs as a symport

of NH3 and H+, where the passage of H+ is coupled to the passage of NH3, leading to a

net uniport of NH4+ (Figure 8B) (Andrade et al., 2005; Andrade & Einsle, 2007). So

far, in order to give more experimental evidence to support this mechanistic model,

research has focused on residues involved in the permeation pathway, including the

external binding site or recruitment site of NH4+, the “Phenylalanine gate”, the

hydrophobic pore and the cytoplasmatic vestibule and possible deprotonation

site(s) (Marini et al., 2006; Javelle et al., 2008; Tremblay & Hallenbeck, 2008;

Lamoureux et al., 2010).

Despite the experimental data obtained so far, the molecular dynamic simulations

and theoretical calculations addressing the question of the transported substrate

and the function of the different conserved amino acids supposed to be involved in

the deprotonation event, it is still controversial how the NH4+ penetrates the

hydrophobic pore and how the conduction of H+ takes place if deprotonation occurs.

Molecular dynamic simulations made in Ec-AmtB proposed that other residues such

as A162 are involved in the coordination of NH4+ during the transition through the

phenylalanine gate (Nygaard et al., 2006; Bostick & Brooks, 2007). Additionally,

these theoretical calculations suggest that the phenylalanine gate is possibly more

permeable to NH4+ than NH3, thus preventing diffusion of NH3 back to the

Introduction

Camila Hernández

25

extracellular side (Lamoureux et al., 2010).

As mentioned, it is so far accepted that Amt proteins bind NH4+ at the extracellular

side of the pore in the recruitment site. However, human Rh proteins lack some of

the key residues involved in the binding of ammonium, suggesting that these

proteins act as NH3 channels (Ripoche et al., 2004; Gruswitz et al., 2010; Mouro-

Chanteloup et al., 2010). In addition, it has been reported by recent structural and

functional studies that the RhCG protein in fact conducts NH3 (Gruswitz et al., 2010).

Recently, Lamoreoux et al. (2010), proposed that co-transport of NH3 and H+ by Amt

proteins is a possible mechanism that might be used by other members of the

ammonium transport family that are known to show electrogenic transport, such as

the Amt1;1 and Amt1;2 from L. esculentum (Ludewig et al., 2002 and 2003) and the

Amt1;1 from A. thaliana (Mayer & Ludewig, 2006; Ludewig, 2006). In this

mechanism based on quantum calculations, they suggest that NH4+ deprotonates

after crossing the phenylalanine gate. At this position, called S2, located at the

entrance of the hydrophobic pore, NH4+ could be bound to residues F215, H168,

W212 (numbered according to Ec-AmtB sequence), and also to either water or

ammonia. In this site, two cation-π interactions are created with residues F215 and

W212, and two strong charge-dipole interactions with H168 and water are formed.

Under this environment, NH4+ could transfer a proton to H168, followed by the

diffusion of NH3 down the pore and the reprotonation of NH3 via H318.

Alternatively, if the excess of protons has already been transferred, reprotonation

takes place in the cytoplasm. The protonation state of the histidine residues can

then be reset via “proton loop” or by side-chain rotation (Lamoureux et al., 2010).

In addition, a second possibility could be conceived, where the H168/H318

interaction provides stabilization of water molecules present in the pore that acts as

a “proton wire” that would allow diffusion of a H+ from NH4+ to the intracellular side

of the pore, followed by the diffusion of NH3 through the hydrophobic pore

(Lamoureux et al., 2010). This proposal clearly supports the involvement of the

highly conserved “twin-his” motif in the transport. So far, however there is no

Introduction

Camila Hernández

26

conclusive experimental evidence for this.

3.3.3 Regulation of Amt proteins

Ammonium uptake and assimilation can be regulated on different levels in the cell.

Upon nitrogen starvation amt genes are highly expressed (von Wirén et al., 2000).

However, in the presence of ammonium, transcription of these genes can be

repressed by nitrogen-regulatory proteins (Arcondéguy et al., 2001). Once Amt

proteins are expressed, regulation of uptake can be achieved through the action of

PII proteins, which control the activity of the transporters and other enzymes

involved (Forchhammer, 2008; Tremblay & Hallenbeck, 2008).

In some prokaryotes, Amt proteins are organized in an operon, which contains a

second gene that encodes for a nitrogen-regulatory protein of the PII family, called

GlnK (product of the glnK gene) (Thomas et al., 2000b). PII proteins are signal

transduction proteins present in archaea, bacteria and plants that can sense

intracellular variations of carbon and nitrogen, and regulate nitrogen assimilation

through protein-protein interactions (Ninfa & Atkinson, 2000; Tremblay &

Hallenbeck, 2008). They are homotrimeric cytoplasmatic proteins, with highly

conserved structures. The trimer presents three protruding loops, called T-loops

(one per monomer), which can exhibit different conformations that are functionally

relevant for signal transduction and importantly involved in protein-protein

interactions (Xu et al., 1998; Sakai et al., 2005; Yildiz et al., 2007).

PII proteins can function in two different modes according to the signal recognition.

A conserved general and basic mode involves the binding of different effector

molecules like ATP, ADP and 2-oxoglutarate (2-OG) (Arcondéguy et al., 2001;

Forchhammer, 2004; Ninfa & Jiang, 2005). PII proteins possess three nucleotide

binding sites located between each subunit (Xu et al., 1998), such that, in the

presence of ATP only, the PII trimer can also bind up to three 2-OG molecules (Ninfa

& Jiang, 2005). In addition, the ATP-binding sites can be competitively occupied by

ADP. However, the presence of 2-OG increases the affinity of these binding sites

Introduction

Camila Hernández

27

towards ATP (Jiang & Ninfa, 2007). The binding of the different effectors influences

the conformation of the T-loop and also the interactions between the PII protein and

the receptor (Yildiz et al., 2007).

A second signal recognition mode not widely conserved involves the covalent

modification of the T-loop. So far, it is known that in proteobacteria, the covalent

modification of the T-loop is used to sense the glutamine levels through an enzyme

called GlnD, which uridylylates a tyrosine residue at the tip of the T-loop (Y51,

numbered as in Ec-GlnK) (Reitzer, 2003; Ninfa & Jiang, 2005). Overall, the covalent

modification at the T-loop and the (cooperative) binding of effector molecules leads

to different conformation states of the PII protein and thus distinct signal

recognition states. Upon these different conformational states, the PII protein can

bind or interact with a variety of PII signal receptors, such as transcription factors,

regulatory enzymes, metabolic enzymes, transport proteins or other proteins

involved in nitrogen metabolism. Complex formation (PII protein- PII signal

receptor) promotes activation or inhibition of activity of the receptor or target

protein (Forchhammer, 2008).

Recently, it has been demonstrated that the uptake of ammonium by Amt proteins is

regulated by GlnK proteins through complex formation (Conroy et al., 2007;

Gruswitz et al., 2007). The interaction between Amt and GlnK is ultimately

determined by the nitrogen requirements of the cell, indicated by the intracellular

pools of glutamine, ATP, ADP and 2-OG (Arcondéguy et al., 2001). The AmtB-GlnK

complex is formed only when nitrogen-deprived cells come across with an increased

nitrogen supply. GlnK proteins are synthesized under nitrogen deprivation; hence,

they accumulate in an uridylylated modified form (Ninfa & Atkinson, 2000). When

the nitrogen supply increases, the uridylylated GlnK protein becomes de-

uridylylated due to a subsequent increment of glutamine levels. Associated with

these events, the levels of 2-OG decrease due to intensifying nitrogen assimilation.

Just when all these conditions converge, GlnK binds to the integral membrane

protein AmtB effectively preventing ammonium transport (Javelle et al., 2004).

The crystal structure of the Ec-AmtB-GlnK complex (Figure 9A) reveals the mode of

Introduction

Camila Hernández

28

interaction between both proteins. Through an extended surface loop (T-loop)

(Figure 9B) that contains a tyrosine residue at the tip (Y51 in Ec-GlnK) GlnK blocks

the cytoplasmic pore exit preventing ammonium translocation. Inhibition by GlnK in

E. coli is then controlled by uridylylation of the Y51 residue preventing complex

formation (Conroy et al., 2007; Gruswitz et al., 2007).

Figure 9: Crystal structure of the Ec-AmtB-GlnK complex. A. Side view of the Ec-AmtB-GlnK

complex, the surface of the AmtB trimer is shown and each monomer is colored in different tones

of blue. The GlnK trimer is located at the C-terminal side and each monomer is shown as a cartoon

representation. B. Close up view of the Ec-GlnK trimer. The protruding T-Loop is indicated for one

monomer. C. Top view of the surface of GlnK that interacts with the AmtB and the threefold axis of

the GlnK trimer. PDB accession code: 2NS1.

Introduction

Camila Hernández

29

3.3.4 Multiplicity of Amt proteins

Often, several copies of amt genes can be found in the genome of one organism. This

is the case with the hyperthermophilic archaeon A. fulgidus where three homologues

of Amt proteins are present. Other organisms like S. cerevisiae (Ludewig et al.,

2001), Methanococcus acetivorans (Galagan J. E., 2002) or the tomato plant,

Lycopersicum sculentum (Ludewig et al., 2002), also have three amt genes within

their genome. In addition, the presence of six copies of amt genes in Arabidopsis

thaliana (Gazzarrini et al., 1999; von Wiren et al., 2000) and even twelve in rice,

Oryza sativa (Bao-zhen et al., 2009) have been reported.

Among the different At-Amt proteins, different substrate affinity rates were

estimated using 14C-labelled methylammonium, indicating different affinities,

transport rates and regulation of the transcription levels of these proteins in

response to the availability of nitrogen supply, photosynthetic products and diurnal

change (Gazzarrini et al., 1999). Consequently, the presence of multiple copies of

these genes may suggest distinct affinities and modes of regulation for Amt proteins.

“Ca. Kuenenia stuttgartiensis” also holds several copies of amt genes. Precisely, five

copies of amt genes (amt1-5) were identified in the genome, located in separate loci.

All five genes present homologies to the amtB and in some cases, for amt1 and amt2,

these genes are followed directly by a gene for a PII nitrogen regulatory protein. The

amt1, amt2 and amt3 genes are present in the same loci. The amt3 however, is

followed by an uncharacterized gene sequence. The amt4 and amt5 genes are

located in two separate loci and present different characteristics than the other amt

genes identified in “Ca. K. stuttgartiensis”. The amt4 encodes for an Amt protein

with the presence of an N-terminal -D-xylosidase domain. The amt5 is transcribed

in the opposite direction to amt1-4. It possesses 2037 base pairs (locus tag:

kuste3690) and encodes for an Amt protein fused with a histidine kinase protein.

This study will be focused on the Ks-Amt5 protein encoded by the amt5 gene which

Introduction

Camila Hernández

30

will be described in the following section.

3.3.5 The Amt protein Ks-Amt5 from “Ca. Kuenenia stuttgartiensis”

The Ks-Amt5 is composed of 679 amino acids with a calculated molecular weight of

74.45 kDa (ProtParam; Gasteiger et al., 2005). A remarkable characteristic of Ks-

Amt5 is the presence of two domains, an N-terminal integral membrane domain and

a C-terminal domain. Based on protein sequence analysis, the protein presents

homologies to Amt proteins for the N-terminal domain (M1-A408) and to a histidine

kinase protein (F413-K679) for the C-terminal domain (Figure 10).

Figure 10: Schematic domain organization of Ks-Amt5.

As typical for the Amt protein family, topology predictions show the presence of

eleven transmembrane helices for the N-terminal domain (Figure 11). The C-

terminal domain referred to as Ks-Kin from here on, contains 266 amino acids with

a calculated molecular weight of 30 kDa (ProtParam; Gasteiger et al., 2005),

constituting approximately one-third of the full-length protein. This domain is

predicted to be entirely cytoplasmatic and possesses a histidine phosphorylation

site and an ATP binding site.

Introduction

Camila Hernández

31

Figure 11: Secondary structure topology prediction for Ks-Amt5. The N-terminus shows

the integral membrane domain (Amt) containing eleven transmembrane helices (I-XI). The

C-terminal region shows the histidine kinase domain located at the intracellular side. The

topology was predicted with TMHMM (Sonnhammer et al., 1998; Krogh et al., 2001) and

schematically plotted with the macro package TEXtopo for Latex (Beizt, 2000).

Despite these interesting features of Ks-Amt5, the localization of the protein is so far

unknown as well as its function within the metabolism of “Ca. Kuenenia

stuttgartiensis”.

3.4 Histidine kinases

Protein phosphorylation is an important process in the regulation of cell function

and a relevant type of post-translational modification of proteins (Stock et al., 2000).

Studies on phosphorylation processes have been specially focused on serine,

threonine and tyrosine phosphorylation. However, histidine phosphorylation also

plays a crucial role in cellular control and regulation especially in prokaryotes

(Besant & Attwood, 2010).

Introduction

Camila Hernández

32

Histidine kinases are signal transduction proteins that control different complex

processes in many organisms. Commonly, histidine kinases are part of the “Two

component signal transduction system” (TCS). TCS are elegant modular systems,

which connect extra-cellular stimuli, such as oxygen or nitrogen levels, to regulatory

events important for adaptation to environmental changes (Klumpp & Krieglstein,

2002). These signal transduction systems are characteristic for prokaryotes

although some studies have mentioned their occurrence in eukaryotes, such as

Arabidopsis and S. cereviseae (Chang et al., 1993; Ota et al., 1993; Maeda et al., 1994).

The most frequent signal transduction mechanism involves two conserved proteins:

a sensor histidine kinase (HK) and an effector response regulator (RR) that are

phosphorylated at a conserved histidine and aspartate residues, respectively

(Casino & Marina, 2009).

The TCS pathway consists mainly of four steps (Figure 12). First, upon a detected

stimulus by a sensor domain of the HK protein an ATP-dependent reaction is carried

out, in which a histidine residue of the HK protein is autophosphorylated.

Subsequently, the phosphoryl group from the phosphohistidine is transferred to an

aspartate residue of a corresponding RR protein. The phosphorylation of the RR

activates an effector domain of the cognate protein that can then interact with

targets, such as genes or other proteins, generating a downstream cellular response.

Finally, the signaling pathway ceases with the dephosphorylation of the RR protein

by an innate or HK-induced autophosphatase activity (Stock et al., 2000; Klumpp &

Krieglstein, 2002).

Introduction

Camila Hernández

33

Figure 12: Two-component signal transduction pathway showing a schematic representation of the

domain organization in histidine kinases and their response regulator proteins. Adapted from Dutta

et al., 1999; Stock et al., 2000; West & Stock, 2001 and Klumpp & Krieglstein, 2002.

3.4.1 Characteristic sequence motifs and function

Histidine kinases exhibit a characteristic modular arquitecture; besides the sensor

region of the protein, the kinase core is constituted by two separate domains, a

Dimerization and Histidine phosphotransfer domain (DHp) and a Catalytic and ATP-

binding domain (CA) (Dutta et al., 1999; Stock et al., 2000; Marina et al., 2005).

Based on amino acid sequence similarity, all histidine kinases additionally present

five unique motifs (boxes), named by their characteristic residues H, N, G1, F and G2,

involved in the binding of ATP and kinase autophosphorylation (Parkinson &

Kofoid, 1992).

The DHp domain includes the H-box, which contains the conserved histidine residue

and thus the site of autophosphorylation. The N, G1, F and G2 boxes are commonly

adjacent to each other and positioned in the CA domain and demarcate the

Introduction

Camila Hernández

34

nucleotide-binding and cleavage site (Stock et al., 2000) (Figure 12). The N-box

contains an asparagine residue and can present a variable length from 5 to 45

residues. The G1 and G2 boxes are glycine-rich portions with DXGXGX and GXGXGX

sequence motifs respectively (Stock et al., 1989). The F-box contains a conserved

phenylalanine residue and it is located between the G1 and G2 boxes (Parkinson &

Kofoid, 1992). Structural and biochemical evidence revealed that HKs function as

dimers, where the mode of autophosphorylation occurs in trans orientation.

Consequently, ATP bound to the CA domain of one monomer transfers its -

phosphoryl group to the histidine residue located in the DHp domain of the other

monomer (Cai & Inouye, 2003; Casino et al., 2009).

Structurally, the H-box is located in a long -hairpin that forms an antiparallel four-

helix bundle with the neighbor DHp domain (Figure 13A). The catalytic domain

presents an --sandwich fold that consists in three -helices and five anti-parallel

-strands (Figure 13B) (Stock et al., 2000). The N and F boxes are located toward

the -strand regions whereas G1 and G2 boxes are forming unstructured segments

or loops. The segment that connects the F and G2 boxes can adopt different

conformations due to its flexibility, thus it is called ATP lid (Bilwes et al., 2001;

Marina et al., 2001). These structural features of the catalytic domain are

homologous to ATPase domains of other proteins like the type II topoisomerase,

Gyrase B, the DNA mismatch repair protein MutL, and the human chaperone Hsp90

(Tanaka et al., 1998; Bilwes et al., 1999).

The different enzymatic activities of HKs (autokinase, phosphotransfer and

phosphatase) entail the contribution of one or both of the DHp and CA domains. This

fact implies the existence of different conformational states of both domains with

respect to one another upon reaction to a certain stimulus (Tanaka et al., 1991;

Hsing et al., 1998). Therefore, recent studies have focused on the structural

characterization of these different states in order to elucidate mechanisms of

reaction and subsequent signaling pathways.

Introduction

Camila Hernández

35

Figure 13: The histidine kinase domains. The nuclear magnetic resonance structure of the A.

dimerization (DHp) domain and B. catalytic (CA) domain of the E. coli osmosensor protein EnvZ.

The DHp domain shows the four-helix bundle formed by two subunits (one colored in silver and

the other colored in blue at the N-terminus and red at the C-terminus). The H-box is labeled

with the conserved histidine residue represented as a stick. The catalytic domain shows an --

sandwich fold where the N, G1, F and G2 boxes are represented in dark blue. The catalytic

domain NMR structure was solved with an ATP analogue (ANPPNP) represented as a stick

model. PDB accession codes: 1JOY and 1BXD respectively.

Additionally to the DHp and the CA domains, HKs present a variety of sensing

domains to detect, directly or indirectly, different environmental signals. These

sensing domains share a low sequence similarity indicating that HKs probably

interact with a certain ligand under specific conditions according to the stimulus

detected (Stock et al., 2000). The sensing domain is located in the N-terminal region

of the protein and it can be a cytosolic or a transmembrane module. The cytosolic

sensing modules can include for instance, a PAS domain (period circadian protein,

aryl hydrocarbon receptor nuclear translocator protein, single-minded protein),

which detects changes in light, redox potential, and small ligands according to their

associated cofactor. HK PAS domains have been studied on the soluble KinA from

Bacillus subtilis (Taylor & Zhulin, 1999) and on the heme-based oxygen sensor, FixL

(Lukat-Rodgers & Rodgers, 1997; Miyatake et al., 1999). PAS domains are frequently

Introduction

Camila Hernández

36

found in HKs and so far several structures of such domains have been solved. In CitA

the PAS domain consists of a five-stranded -sheet and five -helices forming a

central cavity where the ligand, citrate, can bind (Reinelt et al., 2003). Structural

comparison of this domain in the presence and absence of ligand revealed that

citrate binding produces a considerable contraction of the domain. This contraction

was proposed to act as the molecular switch that activates the transmembrane

signaling (Sevvana et al., 2008).

However, in the transmembrane HKs, the sensing domain is attached to the kinase

core through a transmembrane helix and a cytoplasmatic linker. This

transmembrane helix can be variable in length and sequence but it usually includes

a structural element termed HAMP (histidine kinase, adenyl cyclase, methyl-

accepting chemotaxis proteins and phosphatase) or P-type linker (Aravind &

Ponting, 1999; Williams & Steward, 1999). These linkers are variable in length from

40 to 180 residues and present a predicted topology of an -helical, coiled-coil like

motif. Furthermore, it has been suggested that these linkers may be involved in the

transmission of signals between the sensing domain and the kinase core (Fabret et

al., 1999; Williams & Steward, 1999).

3.4.2 Classification of histidine kinase proteins

According to their domain organization, histidine kinases can be separated into two

major classes (Figure 14) (Bilwes et al., 1999). Class I HKs are mostly homodimeric,

where the H-box is directly contiguous to the CA domain and present the above-

mentioned structural organization. Examples of this class are the osmosensor EnvZ

from E. coli (Tanaka et al., 1998; Tomomori et al., 1999) and the sensor histidine-

kinase TM083 from Thermotoga maritima (Marina et al., 2005).

Introduction

Camila Hernández

37

Figure 14: Classification of histidine kinase proteins according to the domain

organization. Class I is represented on top, showing the dimerization domain (DHp)

with the H-box and the ATP-binding domain or catalytic domain (CA) with the N, G1,

F and G2 boxes. Class II (bottom), shows the P1-P5 domains as described for CheA.

Adapted from Dutta et al., 1999; Stock et al., 2000; West & Stock, 2001; and Klumpp

& Krieglstein, 2002.

Class II histidine kinases, exemplified by the chemotaxis protein CheA (Bilwes et al.,

1999), possess five domains P1-P5 from N-terminus to C-terminus. In this type of

kinases, the H-box is located in the His-containing phosphotransfer (Hpt) domain or

P1 domain, which is separate and distinct from the dimerization domain (P3) and

the catalytic domain (P4). The P2 domain, unlike other HKs, is a separate domain

that participates in the recognition and binding of the RRs. Further, the P5 domain is

involved in the interaction of CheA with the chemotaxis receptors and a coupling

protein, CheW (Bilwes et al., 1999; West & Stock, 2001).

Introduction

Camila Hernández

38

3.4.3 Structure of the cytoplasmatic portion of the sensor histidine-kinase TM083 from Thermotoga maritima

As mentioned, Ks-Amt5 exhibits homologies with histidine kinases and in particular

it shares a high degree of similarity with the histidine kinase TM083 from

Thermotoga maritima. The TM083 represented the first crystal structure of the

complete cytoplasmatic region of a sensor histidine kinase revealing previously

unidentified functions for several conserved amino acids and showing for the first

time the disposition of both dimerization and catalytic domains.

The 1.9 Å resolution X-ray crystal structure comprises the cytoplasmatic portion

(residues 233-489) of the sensor histidine kinase TM083. The corresponding

fragment, HK853-CD, confirmed a homodimeric structure with a two-fold symmetry

(Marina et al., 2005). Each HK853-CD subunit contains two domains, an N-terminal

helical-hairpin domain with two anti-parallel helices (1 and 2), and a C-terminal

- domain that contains the characteristic five -strands and three -helices from

the CA domain (Figure 15).

In the overall structure, helices 1 and 2 are connected by a nine-residue turn,

residues S279-T287. The 1 helix, presents a twist induced by a proline residue

(P265) that separates it into two parts, 1a and 1b. The conserved histidine auto-

phosphorylation site is located in 1a (H260), while the 1b helix forms a helix-

bundle with helices 2 and 2’ (symmetry mates).

The catalytic domain exhibits an - sandwich fold with two layers. The first layer is

almost orthogonal to the helical-hairpin domain and includes a mixed five-strands

-sheet formed by strands B, and D-G. The second layer is then formed by three

-helices (3-5). Moreover, some additional components were observed such as a

short pair of anti-parallel strands (A and C) and a disulfide bridge between two

cysteine residues, C330 and C359, that link a segment of helix 3 and the -strand

C.

Introduction

Camila Hernández

39

Figure 15: Crystal structure of the cytoplasmatic portion HK853-CD of the sensor histidine

kinase TM083. A. Dimer representation. One monomer is colored in silver; the second monomer

is colored in rainbow with blue at the N-terminus and red at the C-terminus. B. Details of the

monomer structure, the dimerization (DHp) domain and catalytic (CA) domain are labeled. The

autophosphorylation site shows the conserved histidine residue (His260) represented as a stick

model coordinated by a sulfate ion. The catalytic domain (90° tilt) shows the - sandwich fold.

The protein was crystallized in the presence of an ATP analogue (AMPPNP) that was hydrolyzed to

ADPN represented as a stick. PDB accession code: 2C2A

The knowledge of the HK853-CD structure has given insights into the catalysis and

regulation of class I HKs. As already mentioned, signal transduction pathways begin

with a stimulus that induces a change in the sensor domain of the HK. These

conformational changes are then transferred through the four-helix bundle into the

cytoplasmatic kinase core, hence, influencing the kinase and/or phosphatase

activities carried out by the DHp and CA domains. So far two transduction models

Introduction

Camila Hernández

40

have been described: (1) induced by a rotational movement of the helices with

respect to one another (Cochran & Kim, 1996) and (2) due to a piston-like

movement of one or two helices with respect to the other helices present in the

bundle (Falke & Hazelbauer, 2001). The second transduction model has been more

accepted due to the prevalent evidence from chemotactic receptors. Further, the

linker domains formed by coiled-coil motifs transmit the signal between domains

and possibly modulate and amplify these movements (Marina et al., 2005).

Conclusively, the HK853-CD structure supported the previous knowledge on

histidine classification and catalytic mechanism. However, the major contribution is

the characterization of the first model of interdomain connection between the DHp

and the CA domain of a sensor HK protein.

3.5 Aims of this work

Ks-Amt5 protein is one of the five ammonium transport proteins encoded in the

genome of the anammox bacteria “Candidatus Kuenenia stuttgartiensis”. It presents

remarkable and unusual characteristics that make it an interesting target for

structural biology studies. The presence of two distinct domains, an Amt and a

histidine kinase, identifies it as a novel Amt type and a two component signal-

transduction protein.

The Amt domain of Ks-Amt5 shares characteristic topological similarities with other

Amt proteins. Thus, it is assumed that this domain is likely to preferably form a

stable trimer. The conserved trimeric state of the Amt proteins leads to the question

of how the structural arrangement between the Amt and the HK domain in Ks-Amt5

may look, since so far, histidine kinases have only been described as functioning as

dimers. Besides the structural characteristics, Ks-Amt5 presents a different and not

yet described molecular mechanism by which ammonium sensing could be

integrated as a signal to modulate the phosphorylation state of the histidine kinase

domain as a first step for a signal-transduction pathway. These properties of Ks-

Introduction

Camila Hernández

41

Amt5 may confer a unique biochemical role and function in the metabolism of

“Candidatus Kuenenia stuttgartiensis”.

The aim of this work is to determine the structure of the novel Amt protein, Ks-

Amt5, from the anammox bacteria “Candidatus Kuenenia stuttgartiensis” and

thereby foster comprehension of its ammonium transport mechanism.

Consequently, X-ray crystallography studies were designed to gain insight into the

structural aspects of the transport mechanism. For this, the amt5 gene from “Ca. K.

stuttgartiensis” was cloned and heterologously overexpressed in different E. coli

strains. In addition, different variants of the amt5 have been designed in order to

produce the cytoplasmatic histidine kinase domain of Ks-Amt5. The overproduced

Ks-Amt5 and variants were purified by affinity and size exclusion chromatography.

Subsequently, crystallization trials were carried out to obtain well-diffracting

crystals followed by the determination of the three-dimensional molecular

structure. Moreover, functional studies were performed using different biochemical

methods, such as isothermal titration calorimetry and phosphorylation assays, in

order to relate the activity of both Amt and histidine kinase domains.

Materials and Methods

Camila Hernández

42

4 Materials and Methods

Unless stated otherwise, standard techniques were employed for all experiments.

4.1 Materials

4.1.1 Chemicals

All standard chemicals used were of analytical purity grade (p.a). These chemicals

were obtained from the following companies: Applichem (Darmstadt, Germany), BD

(Heidelberg, Germany) Merck (Darmstadt, Germany), Perkin-Elmer (Rodgau,

Germany), Roth (Karlsruhe, Germany) and Sigma-Aldrich (Deisenhofen, Germany).

4.1.2 Detergents

For the extraction from the membranes and in order to stabilize the membrane

proteins in solution, detergents of very high purity grade were used. These

detergents were purchased from Affymetrix-Anatrace (Maumee, USA).

4.1.3 DNA and Protein Weight Markers

The size of cloned DNA fragments was calculated using 1 kb DNA ladder (MBI

Fermentas, St. Leon-Rot, Germany). For the evaluation of protein size bands in SDS-

PAGE and Western blot membranes, unstained protein molecular weight marker

and Page-RulerTM pre-stained plus protein ladder (MBI Fermentas, St. Leon-Rot,

Germany) were used, respectively.

4.1.4 Enzymes

Enzymes used for molecular biology were obtained from MBI Fermentas (St. Leon-

Rot, Germany), peqlab (Erlangen, Germany), Stratagene (La Jolla, USA), and in the


Camila Hernández

43

case of Taq polymerase, a home-made laboratory stock was used. Table 1 lists the

various enzymes used in this work.

Table 1: Enzymes used for molecular biology work on Ks-Amt5

Enzyme Classification Function and application DpnI Restriction endonuclease Digestion of methylated DNA (mutagenesis) NdeI Restriction endonuclease Digestion of restriction sites (cloning) XhoI Restriction endonuclease Digestion of restriction sites (cloning) KAPA HiFiTM Hot Start Polymerase Synthesis of dsDNA (PCR) Pfu Polymerase Synthesis of dsDNA (PCR) PfuTurbo® Polymerase Synthesis of dsDNA (PCR) Taq Polymerase Synthesis of dsDNA (PCR) T4 DNA Ligase Ligase DNA ligation (cloning)

4.1.5 Bacterial strains

Bacteria are able to incorporate extracellular DNA through their cell walls. This

ability can be artificially enhanced by chemical or electric procedures to produce

competent cells that can multiply or overexpress desired plasmidic constructs. In

this work different E. coli strains were used for this purpose.

E. coli strains XL 10 Gold (Stratagene, USA) and XL 1 Blue (Bullock et al., 1987) were

used for amplification of plasmidic DNA. Heterologous overproduction of Ks-Amt5

and variants was performed using chemically competent E. coli C43 (DE3), a variant

of BL21 (DE3) (Studier & Moffatt, 1986) (Novagen, Darmstadt, Germany), tailored

for the expression of membrane proteins (Miroux & Walker, 1996). E. coli BL21

(DE3) was used for the overproduction of the cytosolic domain (Ks-Kin). Table 2

summarizes the different genotypes of the E. coli strains mentioned above.

Table 2: Genotypes of the different E. coli strains used in this work.

E. coli strain Genotype

XL 10 Gold Tetr∆(mcrA)183 ∆(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac Hte F proAB lacIqZ∆M15 Tn10 (Tetr) Amy Tn5 (Kanr)]

XL1 blue recA1 endAI gyrA96 thi-1 hsdR17 supE44 relA1 lac [F’ proAB lacIqZ∆M15 Tn10 (Tetr)] BL21 (DE3) F- ompT hsdSB (rB- mB-) gal dcm λ (DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5]) C43 (DE3) F- ompT hsdSB (rB- mB-) gal dcm λ (DE3) and two uncharacterized mutations


Camila Hernández

44

4.1.6 DNA oligonucleotides

DNA oligonucleotides were obtained under high purity salt free (HPSF) conditions

from Eurofins MWG Operon (Ebersberg, Germany) and Invitrogen (Darmstadt,

Germany). The identification of cleavable signal peptides in amt5 was carried out

before primer design (Table 3) with SignalP v 3.0 server (Bendtsen et al., 2004) as

well as codon usage variations towards E. coli.

Table 3: Primer sequences for all constructs and variants made in this work. Restriction sites

are highlighted in orange (NdeI) and green (XhoI). Point mutations are shown in blue.

Gene Primer sequence: forward (F) and reverse (R). Length (bp) Tm (˚C) amt5 F: 5’ AGA TAT ACA TAT GGA AAA CAT ACA AAT 3’ 27 54.3

R: 5’ ATT CTC GAG CTT GTT CAC TGG ATT TAT GG 3’ 29 63.9 kin F: 5’ AAA TAC ACA TAT GCT TGA AAA AAG GGT 3’ 28 59.3

R: 5’ ATT CTC GAG CTT GTT CAC TGG ATT TAT GGC 3’ 30 65.4 kin_H460A F: 5’ CAA CAA TGT CAG CTG AGC TGC GCA C 3’ 25 66.3

R: 5’ GTG CGC AGC TCA GCT GAC ATT GTT G 3’ 25 66.3 kin_I612W F: 5’ GAT ACC GGC ATT GGT TGG AAG CCT GAA GAC AAA G 3’ 34 61

R: 5’ CTT TGT CTT CAG GCT TCC AAC CAA TGC CGG TAT C 3’ 34 61 kin_S666W F: 5’ CCT TTG GAA AAG GAT GGA CCT TCT TTT TTA TCT TGC 3’ 36 57

R: 5’ GCA AGA TAA AAA AGA AGG TCC ACT CTT TTC CAA AGG 3’ 36 57 kinS F: 5’ GAC AGC AGA CCT TCA TAT GGC AAA TGT TGC 3’ 30 61.6

R: 5’ GCA ACA TTT GCC ATA TGA AGG TCT GCT GTC 3’ 30 61.6

4.1.7 Plasmids: The pET vector system

All plasmids (pET21a, pET28a and pET15dT) used for the heterologous production

of Ks-Amt5, Ks-Kin and variants in E. coli (4.2.2.4), belong to the pET vector system

(Merck-Novagen, Darmstadt, Germany). These vectors contain a lacI gene that codes

for a lac repressor protein, a T7 promoter specific to T7 RNA polymerase, a lac

operator that can block transcription, a multiple cloning site, a f1 origin of

replication that enables the production of single-stranded DNA under appropriate

conditions and a conventional origin of replication. The expression of the target

gene is controlled by the T7 promoter and the lac operon and can be induced by the

addition of an allolactose-mimicking compound such as isopropyl-β-D-

thiogalactopyranoside (IPTG). The plasmids used differ in the type of protease


Camila Hernández

45

cleavage site, antibiotic resistance and position of the affinity tag. In particular, the

pET21a vector carries a C-terminal His6-Tag sequence and a –lactamase gene that

conveys ampicillin resistance to the host cell and can be used as a selectable marker.

The pET28a vector carries an N-terminal His6-Tag/thrombin/T7-Tag configuration

plus a C-terminal His6-Tag sequence with a kanR gene that transfers kanamycin

resistance to the host cell. The pET15dT vector is a modified version of the pET15b

plasmid. It also contains a –lactamase gene for ampicillin resistance. However, it

carries an N-terminal His10-Tag sequence instead of a His6-tag and an additional TEV

protease cleavage site between the NdeI restriction site and the N-terminal H10-Tag.

The vector charts for pET21a, pET28a and pET15dT are shown in Figure 16.

The gene encoding the full-length wild type protein (Ks-Amt5) was cloned by

GenScript (Piscataway, USA) into the pET21a vector. The portion of the amt5 gene

encoding for the cytosolic domain (Ks-Kin) was cloned into pET28a and pET15dT

vectors. All target genes were introduced into the plasmids through the NdeI and

XhoI restriction sites. Variants of the wild type genes (full length and cytosolic

region) were obtained by site-directed mutagenesis (4.2.1.2) with the original

plasmids.


Camila Hernández

46

Figure 16: Vector charts of the three pET plasmids used for the expression of amt5, kin and

variants. The restriction sites used are indicated in blue, affinity tags are marked in grey and the

protease cleavage sites in magenta. Additional features displayed are: f1 origin of replication,

pBR322 origin of replication, T7 prom (T7 promoter), T7 term (t7 terminator), lacI reg (lac

repressor), lacO reg (lac orperator), amp prom (ampicillin resistance promoter), amp marker

(ampicillin resistance gene) and kan2 marker (kanamycin resistance gene). Plasmid charts were

drawn using PlasMapper (Dong et al., 2004).


Camila Hernández

47

4.2 Methods

4.2.1 Molecular biology

4.2.1.1 Polymerase Chain Reaction (PCR)

The Polymerase Chain Reaction (PCR) was used for the amplification of DNA

fragments as well as for mutagenesis experiments (4.2.1.2). This method is

comprised of three basic steps: denaturation, annealing and extension. In each cycle

of PCR, the three steps are repeated to increase the concentration of the desired

DNA fragments in solution. Initially, the DNA is denatured at a high temperature

(above 90 ˚C) to break the double helix. The denaturation step is followed by the

annealing of the primers that complement and flank the DNA region to be amplified.

At this stage, the temperature is decreased to a value close to the melting

temperature (Tm) of the designed oligonucleotides allowing them to anneal to the

DNA matching sequence providing a starting point for DNA polymerase extension of

the template. Due to the high temperatures used during PCR cycles, the DNA

polymerases need to be stable and for this reason they are obtained from

hyperthermophilic organisms such as Thermus aquaticus (Taq) or Pyrococcus

furiosos (Pfu).

Generally, a PCR mixture contains DNA template (DNA fragment containing the gene

of interest to be amplified), a forward primer, a reverse primer, four desoxy-

ribonucleotides (d TP’s), a polymerase, and a buffer for the polymerase activity.

Table 4 shows the PCR mixtures used for the amplification of the target constructs

and the temperatures for the touchdown PCR program used.


Camila Hernández

48

Table 4: PCR mixture composition and touchdown PCR program

PCR mixture 2 µl Reaction buffer (10X)

0.4 µl dNTPs (10 mM) 0.6 µl Forward primer (10 µM) 0.6 µl Reverse primer (10 µM)

5-100 ng DNA template 0.5 µl DNA polymerase

Add ddH2O to 20 µl Initial stock concentrations shown in brackets.

Touchdown PCR program Step Temperature (˚C) Time (s)

Initial denaturation 98 120 1st cycle (1X) Denaturation 98 30

Primer annealing 70 60 Extension 72 270

2nd cycle (1X) Denaturation 98 30 Primer annealing 66 60 Extension 72 270

3rd cycle (1X) Denaturation 98 30 Primer annealing 63 60 Extension 72 270

4th cycle (1X) Denaturation 98 30 Primer annealing 60 60 Extension 72 270

5th cycle (20X) Denaturation 98 30 Primer annealing 55 60 Extension 72 270

Final extension 72 600 Storage 4-8

The PCR mixture and touchdown temperature cycle program shown in Table 4 were

used to amplify the designed constructs as well as to screen and confirm positive

colonies after ligation (4.2.1.4) into the desire plasmid. The PCR products were

purified using the QIAquick purification kit (Qiagen, Hilden, Germany) and analyzed

by agarose gel electrophoresis (4.2.1.5). The quality of the PCR products was

evaluated by UV-Vis spectrometry. Correct PCR products were sent for sequence

analysis as a final confirmation of the integrity of their DNA sequence (4.2.1.7).


Camila Hernández

49

4.2.1.2 Site-directed mutagenesis

Site-directed mutagenesis is a technique used to modify template DNA, by the

controlled exchange of base pair(s) and deletion or insertion of fragment(s) of DNA.

Primers for mutagenesis must contain the desired mutation in both strand

directions, forward and reverse, and also complement the desired target sequence.

In order to improve annealing to the template DNA the desired mutation is usually

placed in the middle of the primer. To adjust the annealing temperature of the PCR,

the primers melting temperature was calculated using the following formula:

Tm = 81.5 + 0.41(%GC)-675/N-%mismatch

Where N represents the primer length (in bases), %GC stands for the percentage of

guanine and cytosine and %mismatch denotes the percentage of non-aligned bases.

Here, variants of amt5 and kin were made by site-directed mutagenesis using

Quickchange mutagenesis protocol (Stratagene, Cedar Creek, USA).

4.2.1.3 DNA digestion with restriction endonucleases

Restriction endonucleases are present in bacteria and archaea and are found to be

involved in the protection of these organisms from viruses. These enzymes cleave

single stranded or double stranded DNA at a specific sequence called a restriction

site. This recognition sequence is unique for each restriction enzyme and it has

usually has a length of 4-6 nucleotides. Cleavage takes place following the hydrolysis

between two sugar-phosphate backbones of the DNA double helix. Restriction

enzymes can produce blunt or cohesive (sticky) ends, according to their specific

cleavage position. This property is of special interest for molecular biology

techniques such as cloning of a gene into a host vector.

In this work, different restriction enzymes (NdeI, XhoI and DpnI) were used to

specifically digest PCR products for cloning but also for the digestion of methylated,


Camila Hernández

50

parental DNA after site-directed mutagenesis PCR. The typical reaction mixtures

used contained 1 µl of the desired restriction enzyme (10 U.µl-1), 5 µl of reaction

buffer (10X) and 10-30 µl DNA to a final volume of 50 µl. The digestion reaction was

carried out at 37 ˚C for 1-4 hours.

4.2.1.4 DNA ligation

Ligation, in molecular biology, refers to the process where an enzyme, the DNA

ligase, covalently links two ends of DNA or RNA fragments. The ligase joins

fragments by the formation of a phosphodiester bond between the blunt or cohesive

ends of double stranded DNA or RNA. In this study, the T4 ligase (isolated from T4

bacteriophage) was used. The ligation procedure was carried out at 16-22˚C for 16-

18h using the reaction mixture shown in Table 5.

Table 5: Reaction mixture for DNA ligation

2 µl Reaction T4 ligase buffer (10x) 1 µl ATP (50 mM) 1 µl PEG 4000 (50%) 1 µl T4 ligase (5 U. µl-1) * µl Vector pET21a, pET15dT, or pET28a, previously digested with NdeI and XhoI * µl DNA insert, previously digested with NdeI and XhoI

Add ddH2O to20µl * Tested ratios: 10:1; 20:1; 40:1; 100:1 (vector:insert) Initial stocks concentrations are shown in brackets.

4.2.1.5 Agarose gel electrophoresis

Agarose gel electrophoresis is a biochemical technique used to separate mixtures of

DNA or RNA fragments according to size by applying an electric field. Since DNA is

negatively charged due to its phosphate backbone, fragments migrate to the positive

anode through an agarose gel matrix. In principle, separation is based on retention

times relative to the size of the particles: smaller fragments migrate further than

larger fragments throughout the agarose gel. The separation range is thus

determined by the pore sizes of the agarose matrix, which are directly correlated to


Camila Hernández

51

the agarose concentration. Low concentrations of agarose will lead to the formation

of a loose matrix with larger pore sizes, while higher concentrations of agarose will

form a tighter matrix with consequently smaller pores.

Table 6: Composition of the agarose gel electrophoresis buffers

TAE buffer DNA Loading dye (6x) 40 mM Tris-HCl pH 8.0 5% (v/v) Glycerol 20 mM Glacial acetic acid 0.04% (w/v) Bormophenol Blue (BPB) 10 mM EDTA pH 8.0 0.04% (w/v) Xylene Cyanol FF (XCFF)

Here, a 1 % w/v agarose gel in TAE buffer (Table 6) with a separation range

between 400-800 bp was used. Samples were mixed with loading dye (Table 7) and

poured into the gel wells. In addition, DNA molecular weight marker (5 µl in a

separate well) was loaded for size evaluation. Electrophoresis was carried out at 90

V for 1 hour.

After the electrophoresis run, the agarose gel was stained to visualize the separated

DNA fragments. For that, the gel was placed into an ethidium bromide bath

containing 0.5 µg/ml EtBr in TAE buffer for 20-30 min. Ethidium bromide is a

fluorescent dye that intercalates between the base pairs of nucleic acids.

Fluorescence accumulates in the sample bands and can then be detected after

exposure to UV-light at 280 nm. The results were documented using a Gel oc 2000

system (Bio ad, u nchen, Germany) or photographically (Olympus C-3040 3MP).

4.2.1.6 Extraction of DNA from agarose gels

Extraction and recovery of desired DNA (PCR or digestion products) bands from

agarose gels was performed with the ZymoCleanTM DNA Recovery Kit (Zymo

esearch, Irvine, USA) following the manufacturer’s instruction manual.


Camila Hernández

52

4.2.1.7 DNA Sequence Analysis

Confirmation of positives clones or variants of amt5 and kin was performed by DNA

sequence analysis using the T7 forward and reverse primers (Figure 18). For this,

samples were sent to the GATC Biotech AG (Konstanz, Germany). Sequence

chromatograms were analyzed with Chromas (version 2.01, Technelesium Pty, Ltd)

and the resulting forward and reverse sequences aligned with the amt5 gene

sequence for comparison using ClustalW2 (Larkin et al., 2007).

4.2.2 Microbiological methods

4.2.2.1 Escherichia coli cultivation

E. coli strains were cultivated in Luria-Bertani medium (Bertani, 1951). This

medium is composed of 1 % (w/v) tryptone, 0.5 % yeast extract and 1 % sodium

chloride. It was sterilized by autoclaving prior to usage.

Cultures for DNA preparation and pre-cultures for protein production were

incubated overnight at 37˚C. For heterologous expression of the different constructs

in E. coli C43 (DE3) or BL21 (DE3), LB medium was supplemented with 100 µg/ml

ampicillin (pET21a, pET15dt) or 100 µg/ml kanamycin (pET28a). See details in

(4.2.2.4)

4.2.2.2 Production and transformation of E. coli competent cells

Chemically competent cells were prepared under sterile conditions by the

inoculation of a chosen colony of the desired E. coli strain in 500 ml LB medium

supplemented with antibiotics whenever adequate. Cells were grown at 37 ˚C until

OD600= 0.5-0.7. At this point, cells were harvested by centrifugation (10 min at 4000

x g, 4 ˚C). The cell pellet was kept on ice and resuspended in 150 ml cold TBF1 buffer

(Table 7) and chilled for 5 min. After a second centrifugation step (10 min at 4000 x


Camila Hernández

53

g, 4 ˚C) the new cell pellet was finally resuspended in 5 ml cold TBF2 buffer (Table

7) and aliquoted into 50 µl samples that were further frozen in liquid nitrogen and

stored at -80 ˚C.

Table 7: Buffer composition for the production of chemically competent E. coli cells.

TBF1 buffer: pH 5.8 with acetic acid TBF2 buffer: pH 6.5 with 1M NaOH 30 mM Potassium acetate pH 7.0 10 mM NaMOPS pH 7.2 50 mM MnCl2 75 mM CaCl2 10 mM CaCl2 10 mM RbCl

100 mM RbCl 15% (v/v) Glycerol 15% (v/v) Glycerol

In order to incorporate extra-chromosomal DNA plasmids into such chemically

competent cells, a heat shock treatment was performed (Hanahan, 1983). For this, a

50 µl competent cell aliquot was thawed on ice followed by the inoculation of 0.5 µl

of DNA (50-100 ng) under sterile conditions and further incubation on ice for 30

min. The heat shock step was performed at 42 ˚C for 45 sec to enable the passive

permeation of the extra-chromosomal DNA though the cell membrane.

Subsequently, the cells were chilled on ice for 2 min prior to the addition of 300 µl

LB medium and incubated at 37 ˚C for 1 hour with shaking at 750 rpm. The last

incubation step allows the cells to assimilate the inoculated plasmids and the

development of antibiotic resistance. After transformation the cells were inoculated

or plated in selective medium supplemented or not with antibiotics.

4.2.2.3 Plasmid preparation

XL 10 Gold or XL 1Blue E. coli strains were transformed and grown in 5 ml LB

medium supplemented with the respective antibiotic, in order to obtain analytical

amounts of plasmid DNA. Isolation and preparation of plasmidic DNA was then

carried out with the ZyppyTM Plasmid MiniPrep Kit (Zymo Research, Irvine, USA)

according to the instructions manual. However, the elution step was done with 10

m Tris‐HCl pH 8.0 instead of the elution buffer (which contains E TA) included in


Camila Hernández

54

the kit to avoid chelation of the magnesium ions that are required for sequencing

reactions.

Pure plasmid was quantified by UV-absorption at 260 nm with a GeneQuantTM 1300

spectrophotometer (GE Healthcare, Munich, Germany). For that, 1 µl of the isolated

DNA plasmid solution was placed in the TrayCell nanodrop cuvette (Hellma

Analytics, Müllheim, Germany) and measured against the elution buffer (blank).

4.2.2.4 Protein production in E. coli

Expression cultures were made in baffled Erlenmeyer flasks. Therefore, 5-10 ml

pre-culture (4.2.2.1) were inoculated in 500ml LB-Medium (supplemented with the

respective antibiotic) and incubated at 180 rpm. Production of Ks-Amt5 was

induced at 20 ˚C for 18 h while production of Ks-Kin and its variants was achieved at

30 ˚C after 2-3 h of induction.

Induction of expression was performed by adding IPTG to a final concentration of

0.4 mM when the cultures reached an optical density, measured at 600 nm, between

0.5–0.7 Au. For the production of Ks-Amt5, cell cultures were incubated on ice for

10-20 min before IPTG induction (cold induction) after which growth proceeded at

20 ˚C. For each expression culture, generally 18 (1 L) baffled Erlenmeyer flasks

(containing 500 ml LB-medium) were inoculated.

Eventually, cell cultures were harvested by centrifugation at 6000 g (rotor JLA-

8.100) for 15 min at 4°C. The cell pellets were collected and the wet cell mass was

determined. Before storage at -20 ˚C, the samples were shock frozen in liquid

nitrogen.

4.2.3 Protein biochemistry

4.2.3.1 Cell disruption and preparation of purification samples

In order to start the protein purification process, cells containing the over-produced

target protein must be disrupted. Subsequently it is necessary to perform a series of


Camila Hernández

55

centrifugation steps to get rid of undesired cell content and separate the cytosolic

and membrane components. For this, the cell pellets were thawed and resuspended

by constant stirring at 4˚C for 1 h with the addition of 3 ml lysis buffer (20 m Tris-

HCl pH 8.0, 40 mM Imidazole pH 8.0, 500 mM NaCl and plus 10 % v/v glycerol only

for the cells containing over-produced Ks-Amt5) per gram of cells. One pill of EDTA-

free Complete Protease Inhibitor Cocktail (Roche Diagnostics, Basel, Switzerland)

per 50 ml cell suspension was added in order to avoid protease activity and

consequent protein degradation. The homogeneous cell suspension was

mechanically disrupted by passing it four times though a micro fluidizer (M-110P,

Microfluidics, Newton, USA). Disruption occurs due to the high sheer-forces

resulting from the nearly instant and severe pressure differences.

For Ks-Amt5 purification, the broken cell suspension was consequently centrifuged

at 30’000 g for 30 min at 4˚C (rotor: JA-25.50) to separate and eliminate the cell

debris. The supernatant was kept and further ultracentrifuged at 300’000 g for 1 h

at 4˚C (rotor: Ti70) in order to obtain the membrane fraction. The membrane pellet

was carefully resuspended on ice with lysis buffer in a ratio of 10 ml buffer per gram

of wet-membranes. For Ks-Kin purification, the supernatant was readily obtained

after centrifuging the broken cell suspension at 108’800 g for 1 h at 4˚C (rotor: JA-

30.50). The supernatant was used after filtration with a 0.45 nm filter.

4.2.3.2 Solubilization of membranes

Solubilization of membrane proteins involves the use of detergents. Detergents are

lipid-like molecules and as such interact with both hydrophobic and non-

hydrophobic residues. They are essential for the extraction of membrane proteins

from the lipid-bilayer environment. For this purpose, the detergent concentration

used has to be above the critical micelle concentration (CMC), which refers to the

minimum concentration in which molecules of detergents form micelles in solution.

The CMC is temperature-dependent and varies with the salt concentration and pH


Camila Hernández

56

values. Since the CMC is specific for each detergent, solubilization trials have to be

performed to determine the best conditions for the extraction of the maximum

amount possible of target protein. Therefore, to optimize the solubilization step,

different detergent types with variations in size and nature of the hydrophobic

chains as well as the hydrophilic groups must be tested. For successful membrane

protein purification, a detergent has, preferably to be able to, extract the target

protein out of the lipid bilayer and stabilize it. Thus, it must not cause the

denaturation of the protein.

The optimal detergent for the recovery of Ks-Amt5 from the membrane was a non-

ionic and mild detergent, n-dodecyl--D-maltopyranoside (DDM).

Table 8: Chemical characteristics and concentrations of the detergents used in this work. Source: www.affymetrix.com

Detergent name Abb. CMC

(% in H2O) Conc.

used (%) Chemical structure

n-Nonyl-β-D-Maltopyranoside D9M 0.2800 0.650

C21H40O11 (Non-ionic) n-Decyl-β-D-Maltopyranoside D10M 0.0870 0.200

C22H42O11 (Non-ionic) n-Undecyl-β-D-Maltopyranoside D11M 0.0290 0.065

C23H44O11 (Non-ionic) n-Dodecyl-β-D-Maltopyranoside DDM 0.0087 0.030

C24H46O11 (Non-ionic) n-Tridecyl-β-D-Maltopyranoside D13M 0.0017 0.005

C25H48O11 (Non-ionic) n-Octyl-β-D-Glucopyranoside OGP 0.5300 0.800

C14H28O6 (Non-ionic)

n-Dodecyl-N,N-Dimethylamine-N-Oxide

LDAO 0.0230 0.050

C14H31NO (Zwitterionic)

http://www.affymetrix.com/


Camila Hernández

57

In practice, thus, when the membrane fraction (4.2.3.1) was fully resuspended, DDM

was added drop-wise to a final concentration of 1% (w/v), during a continuous slow

stirring at 4˚C. After this, the solution was stirred further at 4˚C for 1h and then

centrifuged at 108’800 g for 45 min, 4˚C (rotor: JA-30-50). This centrifugation step

allows the separation of the solubilized membranes from the insoluble fraction. The

supernatant was kept on ice for further use in affinity chromatography.

4.2.3.3 Affinity chromatography

As a purification technique, affinity chromatography is designed to isolate a

particular target protein. This technique makes use of reversible chemical

interactions, such as ionic or van der Waals-based receptor and ligand binding, to

separate and purify the target from the sample mixture in a chromatographic

matrix. For the purification of recombinant proteins, the most popular affinity

chromatography is IMAC (immobilized metal ion affinity chromatography). IMAC is

based on the covalent interactions of protein residues, especially histidine, to metal

ions, such as nickel, cobalt, or copper. The target protein (with a specific tag) can

then be selectively retained in a chelating resin material (e.g. Ni-Sepharose), which

contains immobilized metal ions. To elute the target protein, different methods can

be used, like changes of pH or by addition of a competitive molecule that can

strongly interact with the resin (e.g. Imidazole).

In this work, a Ni-HisTrap affinity column (HisTrapTM FF Column, GE Healthcare,

Munich, Germany) was used. Ni-HisTrap column retains poly-histidine-tagged

proteins. The affinity of the histidine residues to the Ni2+ ions results in a

coordination complex with the imidazole rings. After the target protein is bound,

contaminants with low and non-specific affinity can be washed with a buffer

containing a low concentration of imidazole (20-40 mM). At higher concentrations,

imidazole competes with the His-tagged protein for the Ni2+ binding sites on the

column leading to the displacement and elution of the target protein from the

column.


Camila Hernández

58

The protocol for the purification of Ks-Amt5 and Ks-Kin is schematized in table 13.

All IMAC purifications were performed at 4˚C using an A KTAprimeTM plus system

(GE Healthcare, Munich, Germany). Solubilized membranes (Ks-Amt5) or soluble

cytosolic fractions (Ks-Kin) were loaded onto a pre-equilibrated 5 ml HisTrap

column with a loading buffer containing 20 mM Tris-HCl pH 8.0, 40 mM Imidazole

pH 8.0, 500 mM NaCl and in the case of Ks-Amt5 an extra 10 % v/v glycerol and a

detergent concentration above the CMC (Table 8). The various proteins were eluted

with an elution buffer that was of the same composition as the loading buffer but

contained a higher imidazole concentration (500 mM). The purification was

followed by the protein absorption at 280 nm. After elution, protein fractions were

pooled and concentrated to 500-1000 µl by ultrafiltration using a Vivaspin 20 ml

concentrator (Sartorius-Stedim Biotech, Go ttingen, Germany) at 4’000 g and 4˚C.

The concentrator’s molecular weight cut off ( WCO) used was according to the

estimated protein size by SDS-PAGE (4.2.3.6), 10 kDa for Ks-Kin and its variants, and

50 kDa for Ks-Amt5 and its variants. The concentrated protein was subsequently

used for size exclusion chromatography. Table 9 shows the standard protocol

employed for the affinity chromatography.

Table 9: Stardard protocol for the purification of Ks-Amt5 and Ks-Kin

Volume (column volume) Step Buffer Fraction size (ml)

Flow rate (ml/min)

6X Equilibration Loading buffer - 2 Amount of sample depending on the purification

Loading of sample Loading buffer 10 0.5-1

6X or until stable baseline Washing #1 Loading buffer - 1-2 6X or until stable baseline Washing #2 5% Elution buffer 5 1-2 3-4X or until stable baseline Elution of protein 50% Elution buffer 3 1-2 5X Washing #3 100% Elution buffer - 2

4.2.3.4 Size exclusion chromatography (SEC)

Size exclusion chromatography (or gel filtration) allows the separation of molecules

according to the difference in their size and it is usually used as the final step in the


Camila Hernández

59

purification of proteins. Applications of this method include the separation of

different oligomeric states of proteins from aggregated proteins, as well as the

estimation of molecular size.

SEC involves the use of chromatographic columns packed with a gel filtration

medium. The medium consists of a porous matrix, which is inert and chemically and

physically stable. Samples are eluted isocratically, without a gradient and, thus, with

the use of a single buffer; this fact renders this technique to be one of the most

straightforward chromatographic methods. Separation occurs according to the

molecular weight as the sample passes through the porous matrix. Smaller

molecules can diffuse into the pores of the gel filtration medium and thus interact

with a larger surface area. This leads to greater retention times for smaller particles.

Any molecules larger than the pore size of the matrix cannot diffuse into the pores

and pass right through the column. As a result, molecules are eluted with decreasing

molecular weight.

To estimate the molecular weight of the target protein samples, a calibration curve

was made for two column models (SuperdexTM 200 10/300 GL and SuperdexTM 200

26/60 HiLoad; GE Healthcare, Munich, Germany) using a HMW Calibration Kit (GE

Healthcare, Fairfield, USA). For this purpose, 50-100 µl of the mixture was injected

onto the column. The mixture contained different proteins of known sizes:

Thyroglobulin (669 kDa), Ferritin (440 kDa), Aldolase (158 kDa),

Conalbumin (75 kDa) and Ovalbumin (43 kDa). The molecular weight can then be

calculated by plotting the logarithm of the molecular weight of the standard

proteins relative to their retention volume on the column and a subsequent linear

regression analysis.

In the present work, SEC was additionally used as a refinement step after the

purification by affinity chromatography to obtain homogeneous samples of the

different proteins (Ks-Amt5 and Ks-Kin) for crystallization experiments. For Ks-

Amt5 the SuperdexTM 200 10/300 GL column was used while for Ks-Kin the

SuperdexTM 200 26/60 HiLoad was used. The columns differ in volume size (23.62


Camila Hernández

60

ml and 300 ml respectively) and were chosen according to their separation

properties and to the volume of sample to inject.

The concentrated proteins previously obtained from the affinity chromatography

step were injected onto the column with the respective SEC buffer (20 mM Tris-HCl

pH 8.0, 100 mM NaCl and for Ks-Amt5 and additional 10 % v/v glycerol plus a

detergent concentration above the CMC, Table 8) at a flow rate of 0.4 ml/min (S200

10/300) or 1 ml/min (S200 26/60). Fractions containing the trimer of Ks-Amt5 and

dimer of Ks-Kin were collected and concentrated to a volume of 200–100 µl in

100,000 and 30,000 MWCO concentrators, respectively. Concentrated protein (Ks-

Amt5 or Ks-Kin) was kept on ice until the concentration was estimated. Samples that

reached high enough concentration levels (5-10 mg/ml) were further used for

crystallization experiments and functionality studies. Afterwards, the samples were

aliquoted in small amounts, frozen in liquid nitrogen and stored at -80 ˚C.

4.2.3.5 Protein concentration determination

Protein concentration was determined by the Bicinchoninic acid assay (BCA) (Smith

et al., 1985). It measures the reduction of Cu2+ to Cu+ in alkaline conditions. The

reduction is caused by the interaction of copper and BCA with peptide bonds and

protein residues like cysteine, tryptophan and tyrosine. The interaction of Cu2+ and

BCA leads to the formation of a green complex that upon reduction of the copper

develops a purple color. The reaction can be followed and quantified by UV-vis

spectroscopy, at a wavelength of 562 nm, which is the maximum of absorption for

the resulting purple complex, the production of which is proportional to the protein

concentration.

The high sensitivity (protein amounts from 0.5 µg/ml), negligible susceptibility to

common buffers or substances, stability, and compatibility with a wide range of

detergents, make BCA a good choice for the determination of protein concentration.

For protein determination, a calibration curve was drawn using various

concentrations of bovine serum albumin (BSA) standards prepared in 100 µl ddH2O


Camila Hernández

61

each (25, 50, 100, 150, 200 and 250 μg/mL). Concentrated protein samples were

diluted with 100 µl ddH2O in different ratios (1:50 and 1:100). For the BCA assay, a

solution of 50:1 ratio BCATM Protein Assay Reagent A (Thermo Fisher Scientific,

Waltham, USA) and 4% (w/v) CuSO4 was prepared. Subsequently, 1ml of this

mixture was added to each BSA standard, protein sample and blank sample

(containing only water). All samples were incubated at 60˚C for 30 min. Absorbance

was measured with a GeneQuantTM 1300 Spectrophotometer (GE Healthcare,

Fairfield, USA) at 562 nm. The protein concentration was calculated according to the

calibration curve. All measurements were performed in duplicate for statistical

accuracy.

4.2.3.6 SDS PAGE electrophoresis

SDS-PAGE stands for Sodium Dodecyl Sulphate PolyAcrylamide Gel Electrophoresis.

It is a widely used technique for the separation of protein mixtures according to

their electrophoretic mobility and for the qualitative analysis of protein samples. It

is commonly used as a purity checkpoint after protein purification.

The use of SDS, a strong anionic detergent, leads to the unfolding and denaturation

of the protein samples by binding to the hydrophobic parts of the protein in a ratio

of 1:1.4 μg protein/μg S S ( eynolds & Tanford, 1970). By doing so, the protein

sample acquires a net negative charge, which is proportional to the length of the

polypeptide chain. Therefore, separation of protein mixtures by SDS-PAGE is

achieved according to their electrophoretic mobility (Laemmli, 1970).

SDS-PAGE gels consist of two parts (a stacking gel and a separating gel), which

characterize the technique as a discontinuous electrophoresis. The polyacrylamide

gel is formed by the polymerization of an acrylamide molecule crosslinked by , ’-

methylene-bisacrylamide (bis-acrylamide). For that, ammonium persulfate (APS)

needs to be added in order to initiate the reaction which is then catalyzed by the

amide base , , ’, ’-tetramethylenediamine (TEMED). Differences in ionic strength


Camila Hernández

62

and pH between the stacking gel and the separating gel, lead to a voltage

discontinuity when a current is applied.

The upper gel, called stacking gel, has a lower percentage of acrylamide (4-5%) and

a low pH, making it less cross-linked with lower ionic strength. The lower ionic

strength creates a high electrical resistance, making low and average molecular

weight proteins (negatively charged by the bound of SDS) migrate faster towards

the separating gel when an electric current is applied. Due to the gradient field

intensity, the protein molecules form a stack according to their electrophoretic

motility. In addition, glycine and Cl- ions from the running buffer contribute to the

stacking effect. The small and motile Cl- ions form a polar running front in the

stacking gel, dampening the effective force of the current for the SDS-enveloped

sample and slowing them down. At the same time, the slower moving zwitterionic

glycine molecules, with a neutral charge at pH 6.8, unshield nearby SDS-protein

samples and increase their mobility.

Once the stacked protein bands reach the frontier of the stacking gel and the

separating gel, the higher degree of polymerization of the separating gel, leads to the

separation of the protein molecules according to their molecular weight.

Table 10: SDS-PAGE stacking gel and separating gel compositions. The percentages of the separating gel were chosen for different separation qualities.

Stacking gel (5%) Separating gel (7.5 %/ 12.5 %)

0.75 ml Stock I 0.5 M Tris-HCL pH 6.8 1.875 ml Stock II 0.5 M Tris-HCL pH 8.8 0.4 % (w/v) SDS 0.4 % (w/v) SDS

0.405 ml Bis-acrylamide (30% w/v) 1.875/3.12 ml Bis-acrylamide (30% w/v) 1.83 ml ddH2O 3.72/2.46 ml ddH2O

15 µl APS (10% w/v) 37.5 µl APS (10% w/v) 3 µl TEMED 3.75 µl TEMED

Initial stock concentrations are braketed

Table 11: Composition of the SDS-PAGE buffers.

5X Loading buffer Running buffer 125 mM Tris-HCl pH 6.8 25 mM Tris

20% (v/v) Glycerol 192 mM Glycine 5% (w/v) SDS 1% (w/v) SDS

0.2% (w/v) BPB 1% (v/v) -mercaptoethanol


Camila Hernández

63

The SDS-PAGE was carried out in a Hoefer miniVE vertical electrophoresis system

(GE Healthcare, Fairfield, USA). The gel solutions (Table 10) were poured into the

SDS-PAGE gel unit and polymerized. For Ks-Kin only 12.5 % separating gels were

used, while for Ks-Amt5 both 7.5 % and 12.5 % resolving gels were used for

phosphorylation assays and analysis of protein purification, respectively. Prior to

loading, the samples were mixed with 5 µl 5X loading buffer (Table 11) and injected

to the gel. In parallel to the samples, 5-7 µl of molecular weight marker was loaded

as a reference to evaluate the size of the resulting protein bands. Generally,

Unstained Protein Ladder (Fermentas/Thermo Fisher Scientific, Waltham, USA) was

used as a marker, although, for SDS gels analyzed by Western blotting, PageRulerTM

Plus Prestained Protein Ladder was used. The SDS ran in a running buffer bath

(Table 11) at a constant current of 45 mA per gel and a voltage of 300 V for

approximately 1 hour. For the visualization of protein bands, SDS gels were

incubated with freshly made Coomassie staining solution (4.2.3.7).

4.2.3.7 Coomasie Brilliant Blue (CBB) staining

Coomassie staining solutions consist of a mixture of two triphenylmethane

compounds, CBB G-250 and CBB R-250. These compounds exhibit unspecific

binding to cationic, hydrophobic and non-polar amino acids, resulting in a protein-

dye complex with an intense blue color that can be visually detected. The Coomassie

staining is commonly used in analytical biochemistry as a staining method for

protein bands on gels after SDS-PAGE electrophoresis (Fazekas de St Groth et al.,

1963; Meyer & Lamberts, 1965).

Table 12: Composition of the Coomassie solutions.

Staining solution Destaining solution 10 % (v/v) Ethanol 10 % (v/v) Ethanol

5 % (v/v) Acetic acid 0.002 % (v/v) CBB (G-250/R-250 4:1)


Camila Hernández

64

After separating the protein samples by SDS-PAGE electrophoresis, gels were

incubated in 50 ml of Coomassie staining solution (Table 12) by continuous shaking

until the molecular weight marker bands appeared. In order to reduce the

background, gels were incubated overnight in 50 ml destaining solution (Table 12),

scanned and documented.

4.2.3.8 Phosphorylation assay

The method used to analyze histidine phosphorylation was based on Marina et al.

(2001). The method is based on the autophosphorylation reaction that occurs when

the kinase protein in question is incubated with ATP. In this reaction, the kinase

covalently incorporates a phosphate group (PO4-) from the ATP to an amino acid

with a free hydroxyl group (in the case of Ks-Amt5, a histidine residue), resulting in

phosphorylated protein and ADP. In order to visualize the autophosphorylation

reaction, radiolabeled ATP-[-32P] (PerkinElmer, Rodgau, Germany) was used. The

isotope phosphorous-32 is widely used in life sciences to label biological molecules,

such as nucleic acids and phosphoproteins. It has a high emission energy (1.7 MeV),

which confers high sensitivity and a half-life of 14.2 days. The beta radiation emitted

by this isotope can be easily detected by liquid scintillation counting or by digital

autoradiography (phosphorimaging).

In this work, phosphorylated protein bands on SDS-PAGE gels were detected by

digital autoradiography. With this technique, radioactive samples (in a gel, filter

paper, or blotting membrane) are exposed to an image plate that contains a thin

layer of a phosphorescent material composed by crystals of barium fluorobromide

and bivalent europium as luminescence center (BaFBr:Eu2+) and protected by a

moisture-proof coating. The ionizing radiation emitted by the samples is absorbed

and stored by the BaFBr:Eu2+ crystals. Throughout the process, the bivalent cation

Eu2+ is oxidized to Eu3+ and the released electron is then trapped in the BaFBr

crystal lattice. After exposure, the image plate is scanned in an imaging system that

uses a helium-neon laser to release the trapped electron from the image plate, thus


Camila Hernández

65

reducing Eu3+ to Eu2+. The stored energy is re-emitted in the form of blue light,

which is detected by a photomultiplier. The intensity of the blue light is proportional

to the amount of radioactivity in the sample; thus, the data is stored as a digital

image that contains the locations and intensities of the radioactivity in the samples.

Furthermore, the resulting digital image can be analyzed by image analysis

software, which allows the quantification of signal intensity differences.

Samples of Ks-Amt5 and Ks-Kin were incubated with radioactive ATP-[-32P] (10-50

µCi at >5000 Ci/mmol specific activity) (PerkinElmer, Rodgau, Germany) for 1 h at

30˚C. The working buffer was composed of 20 mM Tris-HCl pH 8.0, 100 mM NaCl, 50

mM MgCl2, and 50 mM MnCl2; in the case of Ks-Amt5 10 % of glycerol was also

added. Additionally, the effect of different concentrations of non-radioactive ATP,

magnesium chloride, manganese chloride or ammonium chloride as well as times of

reactions were evaluated. The standard mixtures for the phosphorylation reaction

are shown in Table 13.

Table 13: Standard phosphorylation reaction mixtures.

For Ks-Amt5 For Ks-Kin 10 µl ATP-[-32P] (250 µCi) 2 µl ATP-[-32P] (250 µCi)

2 µl Non-radioactive ATP (10 mM) 2 µl Non-radioactive ATP (10 mM) 30 µM Ks-Amt5 10 µM Ks-Kin 1-5 µl NH4Cl (10 mM – 2 M)

Reactions were prepared to a 20 µl final volume Initial stock concentrations are bracketed

After the reaction, the samples were mixed with 7 µl SDS-PAGE sample buffer and

loaded into an SDS gel and subjected to SDS-PAGE (4.2.3.6), with a separating gel of

7.5% for Ks-Amt5 and 12.5% for Ks-Kin. Subsequently, the gels were fixed with a

solution of 40% methanol and 20% acetic acid for 5-10 min and were dried in a

BioRad Gel Dryer (BioRad,) for 45-60 min. Bands of phosphorylated protein were

detected by digital autoradiography with a StormTM imager system (GE healthcare,

Munich, Germany) and documented.


Camila Hernández

66

All radioactive experiments were carried out in the Department of Prof. Dr. Nikolaus

Pfanner at the Institute of Biochemistry and Molecular Biology, University of

Freiburg.

4.2.3.9 Western blot

The Western blot (Burnette, 1981) or protein immunoblotting is a common and

highly specific analytical technique used to identify or localize specific proteins from

a mixture sample by the use of antibodies. The protein sample is first separated by

SDS-PAGE electrophoresis. Afterwards, the protein bands can be transferred from

the gel onto a membrane made of nitrocellulose or polyvinyliden difluoride (PVDF),

in order to detect the target protein specifically (Renart et al., 1979; Towbin et al.,

1979). The transfer can be achieved by capillarity action, bringing the protein

solution into the membrane or by applying an electric current, which drags the

protein to the membrane. After the transfer is completed, the membrane is blocked

with a protein-rich solution such as BSA, casein or milk to saturate the free binding

spaces on the membrane and to avoid unspecific interactions of the antibodies.

Detection is carried out by exposing the membrane to antibodies that specifically

recognize and bind to the target protein, either by a specific motive or by an affinity

tag within the protein. A first antibody binds directly and specifically to the target

protein. A secondary antibody, linked to a reporter enzyme carrying alkaline

phosphatase or peroxidase activity, binds to the antigenic primary antibody. Once

the secondary antibody is bound, the reporter enzyme can convert a substrate

soluble dye into an insoluble dye that precipitates onto the membrane so that the

bands, which contain the protein target, can be stained.

This technique was used to identify His-tagged proteins (Ks-Amt5 and Ks-Kin) by

the use of a tetra-his antibody (as a primary antibody) and an alkaline phosphatase

conjugated antibody (as a secondary antibody). For the detection of the protein

bands, the substrates for the alkaline phosphatase conjugated antibody, 5-Bromo-4-

Chloro-3-indolyl phosphate (BCIP) and Nitro-blue tetrazodium (NBT) were used as


Camila Hernández

67

staining reagents. The results were documented by scanning the stained

membranes. Tables 14 and 15 show the solutions used.

The following protocol for blotting was used. The starting point of the blotting

protocol is the activation of the PDVF membrane by incubation in methanol for 5

min. Both the gel, after SDS-PAGE (4.2.3.6), and the PDVF membrane were

equilibrated with transfer buffer by incubating them separately twice for 5 min.

The equilibrated gel and PDVF membrane were placed into a wet blotting system

(miniVE System Blot Module, GE Healthcare) filled with transfer buffer.

Electroblotting was carried out at 25 V for 1.5-2 h. Once the transfer was completed,

the membrane was incubated with blocking buffer for 1 h by continuous shaking.

After the blocking step, the membrane was rinsed 3 times with TBS T/T buffer and

incubated with the primary antibody (mouse anti-4His antibody 1:2000 in blocking

buffer) over night at 4˚C. In a subsequent washing step, the membrane was rinsed 3

times with TBS T/T buffer, incubated with blocking buffer for 5 min during

continuous shaking, and rinsed again 3 times with TBS T/T. These steps were

repeated after 1 hour incubation with the secondary antibody (goat anti-mouse

Alkaline Phosphatase conjugated antibody 1:10000 in blocking buffer). Detection

Table 14: Compositions of the Western blot buffers.

Transfer buffer TBS Tween/Triton buffer (TBS T/T) Blocking buffer 25 mM Tris-HCl pH 7.5 20 mM Tris-HCl pH 7.5 5% (w/v) dry skim milk

in TBS T/T buffer 192 mM Glycine 0.5 M NaCl

0.1% (w/v) SDS 0.05% (v/v) Tween 20

20% (v/v) Methanol 0.1% (v/v) Triton X-100

Table 15: Compositions of the Western blot staining solutions.

Staining buffer Staining solution (per PDVF membrane)

100 mM Tris-HCl pH 9.5 10 ml Staining buffer

100 mM NaCl 33 µl BCiP (5% BCiP in 100% dimethylformamide)

5mM MgCl2 66 µl NBT (5% NBT in 70% dimethylformamide)


Camila Hernández

68

was performed by incubation of the PBDF membrane in staining solution until the

protein bands or a faint background appeared. To stop the reaction, the membrane

was immediately rinsed with ddH2O to avoid further overexposure to reagents.

4.2.3.10 Blue Native PAGE (BN-PAGE)

Similar to the SDS-PAGE, the BN-PAGE is based on a polymerized acrylamide matrix

that separates proteins according to their molecular size. However, BN-PAGE does

not denature the protein sample, but uses instead the Coomassie Brilliant Blue dye

to provide the negative charges to the protein complexes to allow separation by

electrophoresis (Scha gger & von Jagow, 1991; Wittig et al., 2006). Therefore, this

technique can be used to isolate protein complexes, identify protein-protein

interactions and to determine native protein size and oligomeric states.

In this work, BN-PAGE was used to confirm the SEC results regarding the molecular

size and oligomeric state of the Ks-Amt5 protein. The protocol used was made from

a combination of two different protocols ( o gtle et al., 2010; Wittig et al., 2006) and

was adapted to the Hoefer miniVE vertical electrophoresis system (GE Healthcare,

Fairfield, USA).

Samples of Ks-Amt5 (8µl of 5-20µg protein + 2µl 5X loading dye, Table 16) were

loaded onto a 9% (v/v) acrylamide separating gel cast with a 4% (v/v) acrylamide

stacking gel (gel compositions in Table 17). Native Mark (Invitrogen, Carlsbad, USA)

and the HMW Native Marker (GE Healthcare, Fairfield, USA) were used as molecular

weight standards. BN-PAGE was carried out at 4 ˚C (buffers in Table 18). The gel

was run at 100 V for the first 15 min and at 15 mA for 2-4 hours. After

electrophoresis, the gel was fixed and destained overnight in fixing solution (Table

19). Finally, the gel was stained using the CBB protocol for SDS-PAGE (4.2.3.7).

Table 16: Composition of the gel buffer and loading dye for BN-PAGE.

3X gel buffer (pH 7.0) Loading dye 150 mM Bis-Tris/HCl 0.05% (w/v) Ponceau S 220 mM -Amino n-caproic acid 25% (w/v) Glycerol


Camila Hernández

69

Table 17: Composition of the BN-PAGE gels (initial stocks concentrations are bracketed).

Stacking gel (4%) Separating gel (9%) 0.4 ml 30 % acrylamide with 8% (w/v) bis-

acrylamide 2.7 ml 30 % acrylamide with 8% (w/v) bis-

acrylamide 1 ml 3X gel buffer 3 ml 3X gel buffer

1.6 ml ddH2O to a final vol. 3ml 1.8 ml Glycerol 100% (v/v) 20 µl APS (10% w/v) 1.5 ml ddH2O to a final vol. 9ml

2 µl TEMED 30 µl APS (10% w/v) 3 µl TEMED

Table 19: Composition of the Fixing solution for BN-PAGE.

4.2.3.11 Isothermal titration calorimetry

Isothermal tritation calorimetry (ITC) is a thermodynamic technique used to

measure biomolecular interactions, such as protein-protein or protein-ligand

interactions (Pierce et al., 1999). ITC can directly measure the heat released and

absorbed due to a binding event. This allows the determination of binding

parameters such as binding affinity constant (Ka), reaction stoichiometry (n),

enthalpy changes (H) and entropy changes (S). Thereafter, Gibbs energy changes

(G) can be calculated according to the following relation:

Table 18: Buffer compositions for BN-PAGE.

Cathode buffer (10X) pH 7.0 (upper buffer) Anode buffer (10X) (lower buffer) 500 mM Tricine 500 mM Bis-Tris/HCl pH 7.0 150 mM Bis-Tris/HCl

0.2% (w/v) Coomassie G250

Fixing solution 50% (v/v) Methanol 10% (v/v) Acetic acid

100 mM Ammonium acetate


Camila Hernández

70

As a result, an ITC experiment provides thermodynamic information on molecular

interactions that are useful in elucidating function and mechanisms of complex

formation or protein-ligand binding.

In an ITC experiment, a solution of one type of biomolecule (ligand) is titrated into a

second solution of a different biomolecule (binding partner) at a precise and

constant temperature. If the macromolecules interact, the heat (H) that is absorbed

or released is measured over time. H stands in a direct relation to the grade of

binding. When the system reaches saturation, the heat signal decreases until the

background heat of dilution is observed. Consequently, a binding curve is obtained

from the measured heat of every injection against the ratio of ligand and binding

partner. Subsequently, upon analysis of the binding curve with an appropriate

binding model, the thermodynamic parameters mentioned above can be

determined. Figure 17 shows a schematic representation of an ITC instrument.

For an ITC experiment, the precision of the initial concentrations of ligand and

binding partner are important; therefore, they have to be determined with a high

accuracy. Other parameters that need to be considered before running an ITC

experiment are the injection number and volume. A unitless value c (Wiseman et al.,

1989) can be used to choose the optimal conditions for the experiment. This c value

is the product of the binding constant Ka, the initial concentration of the

macromolecule M, and the stoichiometry of the reaction, n:

The value of c defines the shape of the binding isotherm. High c values prevent the

determination of the binding constant Ka due to the fact that it would indicate a very

sudden transition between the no saturation state and the saturation state. As a

result, only a few points define the expected binding curve that would in fact exhibit

a rectangular shape. On the other hand, at low c values (c ≤ 0.1) the isotherm loses

the characteristic sigmoidal shape and reaches linearity due to the wide transitions


Camila Hernández

71

between the turning points. Hence, the determination of the binding constant and

the enthalpy changes becomes inaccurate. Nevertheless, the c value can be changed

by modifying the concentration of the titrant solution. For an optimal determination

of the binding constant, the c value should be between 1 and 1000 (Wiseman et al.,

1989).

Figure 17: Scheme of the VP-ITC device (GE Healthcare). The protein solution is loaded into the

sample cell while the ligand or titrant is loaded into the syringe. During titration, the syringe rotates

in place to stir the solution and the plunger (computer-controlled) injects precise volumes of ligand.

The reference cell is kept at a constant temperature. Temperature differences between the reference

cell and the sample cell (T1) are measured during the experiment and a feedback power (or

differential power) is applied to maintain both cells at the same temperature. For that, a second

temperature difference (T2) between the cells and the inner shield (adiabatic jacket) is measured.


Camila Hernández

72

Moreover, the selection of buffers is also important in an ITC experiment. In a

complex formation, protons can be caught or liberated, thus an equivalent number

of protons will be also caught or liberated by the buffer. In order to determine

accurately the enthalpy changes due to the molecular interaction, both ligand and

binding partner solution should be prepared in the same buffer to reduce errors

deriving from dissimilar buffer components.

Additionally, the number, volume and time length of the injections are important

parameters for the quality of the data. They define the baseline region for the

determination of the enthalpy of binding and the equivalence region given by the

concentration range to determine the binding constant. Therefore, it is also

important that the concentration range selected allows an equilibrium between

measurable amounts of free and bound ligand within the titration zone, which is

defined by the titrant injections.

4.2.3.11.1 ITC experiments with Ks-Kin

In order to determine the binding parameters for Ks-Kin all ITC experiments were

performed at 20 ˚C using the P-ITC microcalorimeter (GE Healthcare, Munich,

Germany). The protein (Ks-Kin) and ligands (ATP, ATPS, ADP, GTP) were prepared

in working buffer containing 50 mM Tris-HCl pH 8.0, 100 mM NaCl and 1 mM MgCl2.

The protein concentration used was 100 µM with a ligand stock initial concentration

of 1.3 mM. Before measuring, all solutions were degassed and brought to working

temperature. Additionally, the sample cell and the injection syringe were cleaned

with water and equilibrated with working buffer. For all experiments, 1.4 ml of

protein solution was filled into the sample cell avoiding the formation of air bubbles

and the ligand solution was loaded into the injection syringe (282 µl).

Titrations were run with 21 injections, a first injection of 2 µl and the remaining

twenty of 14 µl each. The data point given by the first injection was removed from

the resulting data before the curve-fitting. The initial injection is generally inexact


Camila Hernández

73

due to the interval of time that the injection syringe is in the cell during temperature

equilibration. The blank or reference titrations were performed under the same

conditions, however without protein in the sample cell. The resulted heat of dilution

was subtracted from the experimental data during data evaluation.

All obtained data were processed with the program Origin 7.0 using one set of

binding site model to fit the data and the standard Levenberg-Marquardt algorithm

(Levenberg, 1944; Marquardt, 1963).

4.3 Protein crystallography

4.3.1 Crystallization

The determination of three-dimensional protein structures by X-ray diffraction

experiments requires well-ordered crystals of the protein of interest. Crystallization

is a thermodynamically favored process, in which molecules in solution form a

three-dimensional solid block with high long-range order: crystals. The process of

crystal formation is driven by the loss of the ordered hydration shell from the

protein molecules, which leads to a gain of entropy in the system.

Several techniques exist to promote protein crystallization. Vapor diffusion is the

most common and widely used method to obtain protein crystals (McPherson,

1982). In this method, the protein solution is lead to supersaturation by the

equilibration of a drop containing a mixture of protein and a precipitant solution

through a gas phase against the reservoir solution in a closed environment (Figure

18A). Equilibrium between the drop and the reservoir is reached by the gradual

evaporation of water in the droplet. This leads to an increase in the concentration of

protein and precipitant. If the supersaturation of the sample is too high, the

precipitation phase is reached, resulting in aggregates of the protein sample in the

drop. Ideally, the protein solution should reach a nucleation state during the

supersaturation process while the precipitant concentration increases to a suitable


Camila Hernández

74

level to allow the formation of crystalline nuclei and therefore crystallization. The

nucleation state is required for crystal growth (Figure 18B).

Figure 18: Protein crystallization technique and phase diagram. A. Schematic representation of

the sitting-drop vapor diffusion method. In a closed environment, a drop containing a mixture of

protein and reservoir (precipitant) solution equilibrates through the evaporation of water from the

drop, with a reservoir solution. In this process, the concentration of protein and precipitant increase

until equilibrium is reached. Under optimal conditions protein crystallization can occur. B. Protein

crystallization phase diagram. Nucleation in the supersaturation zone is essential for crystal

formation (grey). Crystal growth occurs after the formation of the nuclei (dark blue). Under

unsaturated (white) or precipitation (light blue) phases the crystallization process cannot occur.

4.3.2 Crystallization of Ks-Amt5

For all crystallization trials, the sitting drop vapor diffusion technique was used.

Crystallization experiments were performed manually or using the OryxNano

Protein Crystallization Robot (Douglas Instruments, East Garston, UK). Generally,

0.3-1 µl of protein solution with concentrations from 4-20 mg/ml were mixed with

0.3-1 µl of reservoir solution (50-300µl). Commercially available screens from


Camila Hernández

75

different companies (Hampton research, Aliso Viejo, USA; Jena Biosciences, Jena,

Germany) as well as laboratory-made screens Membrane screen (Stura, 1999),

Footprint I-III (Stura et al., 1992, Stura et al., 1994) were used for initial sparse

matrix sampling. As crystallization plates, 96-well sitting drop Intelli-plates (Art

Robbins Instruments, Sunnyvale, USA) and 24-well sitting drop Cryschem 24-1 SBS

plates (Hampton Research, Aliso Viejo, USA) were used. After the setup, the sealed

crystallization plates were stored at a constant temperature (4 ˚C, 20 ˚C, 25 ˚C, 30 ˚C

or 37 ˚C). The crystallization process was followed by observation via microscope at

regular time intervals and documented.

4.3.3 Finescreens

As initial crystals appeared, optimization of the initial condition was achieved by

varying parameters such as protein concentration, pH, buffer type, salt or

precipitant concentration. Additionally, different drop sizes and protein-reservoir

ratios were tested. These optimization screens, finescreens, were carried out to

improve size and quality of the crystals and were usually designed for 24-well

sitting drop Cryschem 24‐1 SBS plates (Hampton esearch, Aliso iejo, USA).

Co-crystallization experiments were also performed with ATP and non-hydrolyzable

ATP analogues, such as AppCp, ATPS or ATPS, by adding these solutions directly

to the protein-reservoir drop or by incubation of the protein with these solutions to

a final concentration from 0.1-2 mM. In addition to the co-crystallization

experiments, already obtained crystals of Ks-Amt5 were immersed for different

incubation times (1-5 min) to a mixture of the corresponding reservoir solution and

ATP or non-hydrolyzable ATP analogues to a final concentration of 1 mM.

Soaking experiments with Ks-Amt5 crystals were also performed with heavy atoms

solutions. For this 0.2 µl of the heavy atom solution was added to a crystallization

drop where crystals were observed. The crystallization plates were further sealed

and incubated at a constant temperature (20 ˚C) for 24 to 48 hours. Crystals in these

conditions were tested for MAD experiments.


Camila Hernández

76

4.3.4 Structure determination by X-ray crystallography

The determination of protein structures on an atomic level requires a high-

resolution technique that can analyze atomic distances between single atoms, such

as, C, H, N, O, S covalent bonds in the range of 1.0-1.8 Å, or more precisely carbon-

carbon -bonds (1.54 Å). X-rays are a type of electromagnetic radiation with a

wavelength range between 0.1-100 Å. This wavelength interval lies within the

proper spectral range to resolve macromolecular structures. However, it is of

relevance that the intensity of diffraction by a single protein molecule is too low to

be detected. Therefore, for protein structure determination by X-ray diffraction

experiments crystals are needed. The molecules forming crystals are highly ordered

in a regular lattice, this three-dimensional arrangement produces an enhanced

diffraction by means of a constructive interference of the diffracted photons whose

intensities can be then detectable and measured in an X-ray detection setup.

4.3.5 Crystal arrangement

Crystals are three-dimensional blocks of regular repeats of molecules. This regular

and systematic order of atoms is defined as a crystal lattice. The smallest repeating

component of a crystal that forms the whole lattice by translation is the unit cell. It

describes the arrangement of atoms in the crystal by its lattice parameters that

consist of the lengths of the cell edges (constants: a, b, c) and the angles between

them (, , ). The most basic structural element of the unit cell is the asymmetric

unit, which can be rotated and translated to form the whole content of the unit cell

using crystallographic symmetry operators.

The geometry and symmetry of the unit cell defines the space group of the crystal.

For chiral molecules such as proteins, only 65 enantiomorphic space groups are

suitable. These space groups are distributed in seven crystal systems: triclinic,

monoclinic, tetragonal, trigonal, hexagonal and cubic. The correct identification of

the space group is essential for the interpretation of the diffraction data obtained by


Camila Hernández

77

X-ray diffraction experiments and, therefore, for the determination of the protein

crystal structure.

4.3.6 X-ray diffraction by protein crystals

X-rays described as electromagnetic waves, interact with the atoms in a crystal,

especially with the atom’s electrons. This interaction causes the scattering of the X-

ray waves. In case of X-ray beams, incoherent and coherent scattering occurs.

Incoherent scattering happens if an electron interacts with its atom producing

transitions that result in emission of photons of lower energy which eventually

leads to radiation damage. Under coherent scattering, an X-ray photon induces the

oscillation of an atom’s electron with the same frequency as the incident radiation

leading the electron to emit radiation in a random direction but with the same

frequency as the incident X-ray beam. The re-emitted waves can experience a

physical phenomenon called interference. When single scattered X-ray waves have a

phase shift of 180˚, they subtract from each other causing destructive interference.

However, when the scattered waves have the same phase, they can overlap adding

to each other and producing constructive interference. The constructive

interferences produce a diffraction pattern that is fulfilled by the correct orientation

and corresponding positions of all electrons in the unit cells. Further, the diffracted

X-rays on the real crystal lattice create another three-dimensional lattice of

diffraction maxima with inverse geometric properties called a reciprocal lattice

(Figure 19A).

The diffraction of electrons in a crystal lattice that undergo constructive

interference is known as Bragg diffraction and it follows the condition given by the

Bragg’s Law (Bragg, 1913) (Figure 19B):


Camila Hernández

78

Where n is an integer, λ is the wavelength of the incident X-ray beam, d is the

distance between the scattering lattice planes identified by the Miller indices h, k, l

and θ is the angle of the incident wave with respect to the lattice plane. Thus, the

specific directions identified by Bragg’s Law appearing as spots on a diffraction

pattern are called reflections.

Figure 19: Reciprocal lattice planes and Bragg’s law. A. Lattice planes that divide the unit cell

sides into a number of integer fractions allow constructive interference of diffracted waves. The

number of fractions is used to index the planes. In the representation above, the set of planes have

the Miller indices (4 2 3) B. Graphic representation of the Bragg’s law. Two waves reflected by two

adjacent and parallel lattice planes with distance d have a difference in path length of 2dsin.

Constructive interference occurs when this difference in path length is an integer n multiple of the

wavelength used: 2d sin = n.

A geometric tool that demonstrates the relation between the wave vector of the

incident and diffracted X-ray beams, the diffraction angle for a given reflection and

the reciprocal lattice of the crystal, according to Bragg’s law is known as the Ewald

sphere and it is used in X-ray crystallography to construct the reciprocal lattice

points (Ewald, 1969).

The Ewald sphere (Figure 20) is constructed with a radius of 1/λ that passes

through the origin O of the reciprocal lattice considering the crystal in its centre.

The origin of the reciprocal lattice (0,0,0) lies in the transmitted beam at the edge of


Camila Hernández

79

the sphere and opposite to the point S0 where the incident beam enters the sphere.

Thus, the Ewald sphere represents in reciprocal space, all the possible points where

reflections satisfy Bragg’s law.

Figure 20: The Ewald sphere. In reciprocal space, a crystal (C) (orange) is placed in the center of

the Ewald sphere, in this two-dimensional representation, a circle, with a radius of 1/. The entrance

of an incident beam (blue arrows) S0 is opposite to the origin of the reciprocal lattice O. For a given

orientation of the crystal and the corresponding reciprocal lattice (blue points), diffraction

conditions will be satisfied by the reciprocal lattice points that intersect the Ewald sphere (purple

points). The rotation of the crystal and thus the rotation of the reciprocal lattice will lead to the

intersection of different reciprocal lattice points with the Ewald sphere and therefore more

diffraction spots.

The rotation of the crystal implies the rotation of the reciprocal lattice. Therefore, by

every rotation, different reciprocal lattice points intersect the sphere giving a

detectable reflection that represents one lattice plane (h, k, l). These diffraction

spots can be then recorded on an X-ray detector.


Camila Hernández

80

The measurement of the position and intensity of every diffraction spot or reflection

(h,k,l) gives the primary knowledge of an X-ray data collection experiment. This

information is then used to infer the geometry of the crystal, the content and

dimensions of the unit cell and the space group.

4.3.7 The electron density function

The result of a crystallographic experiment is a map of the distribution of electrons

in a molecule, an electron density map. Electrons are generally tightly localized

around the nuclei of the atoms; therefore, an electron density map gives a good

picture of the molecule structure.

The scattering amplitude of the X-ray waves by an isolated atom is measured by the

atomic form factor, or atomic scattering factor. Due to the different number of

electrons present in distinct atoms, the atomic scattering factor increases with the

atomic number, Z, of the atoms in a molecule and thus varies for each element of the

periodic table. Assuming a spherically symmetric distribution of the electron shell,

the atomic scattering factor (f0) is defined as:

The atomic scattering factor is independent on the direction of the incident beam.

However, the phase difference for photons diffracted at different positions in the

electron shell increases with the diffraction angle that is directly coupled via

Bragg’s law to the resolution. Hence, at higher diffraction angles the increase in the

phase difference due to the atoms size will lead to destructive interference, thus,

limiting the diffraction power.

Another attenuation of the X-ray scattering with the increase of the angle is caused

by the thermal motion of the atoms. The factor that describes these attenuations is

referred as the Debye-Waller temperature factor or B-factor. This factor is

incorporated to the atomic scattering factor as an additional exponential term:


Camila Hernández

81

For protein structures, the B-factors can be interpreted as indicators of the

flexibility of different parts of the structure, hence, atoms with low B-factors belong

to the well-ordered parts and atoms with high B-factors exhibit a higher vibrational

motion thus belonging to the flexible parts of the structure.

Every atom in the unit cell contributes to every single reflection (h,k,l) according to

its chemical properties and to its relative position. Therefore, the scattering of all

atoms in the asymmetric unit is the sum of all atomic scattering factors (fj). Thus this

summation is a function of all atoms in the unit cell, known as the structure

factor Fhkl:

Further, the structure factor Fhkl is a wave function that can be divided into two

parts, the amplitude |Fhkl| and the phase angle ei(hkl).

The reciprocal lattice is a Fourier transform and a sum of the structure factors

representing the electron distribution in the crystal. Thus, by means of an inverse

Fourier transformation the electron density function ρ(x,y,z) can be determined for

every point (x,y,z) in real space:

The electron density function is then formed by the sum of all reflection amplitudes

and phases. However in an X-ray experiment, only the intensity of the reflections


Camila Hernández

82

can be measured. The intensity for every reflection (h,k,l) is proportional to the

square of the amplitudes:

The structure factor amplitudes can be derived from the intensities measured as

indicated. However, the information regarding the phase angle is lost during the X-

ray experiment. As a result, without the correct phase angles, the electron density

function cannot be directly estimated. This issue is referred to as the “phase

problem” of crystallography.

Nevertheless, different experimental methods have been developed to indirectly

obtain the phase information for macromolecules: molecular replacement (MR)

(Rossmann & Blow, 1962; Huber, 1965); single/multiple isomorphous replacement

(SIR/MIR) (Green et al., 1954; Perutz, 1956); single/multiple anomalous dispersion

(SAD/MAD) (Hendrickson et al., 1988; Dauter et al., 2002; Dodson, 2003);

combination of isomorphous replacement and anomalous dispersion:

single/multiple isomorphous replacement with anomalous scattering

(SIRAS/MIRAS); radiation induced phasing (RIP) (Banumathi et al., 2004) and direct

methods. In this study, the phase information for the Ks-Amt5 structural model was

obtained by MR.

4.3.8 Molecular replacement

Molecular replacement is one of the techniques used for the determination of

protein structures and frequently applied to the solution of the phase problem. It

requires a previously solved homologous structural model, from which the phases

can be derived. This phase information is then used as the initial phases for the

unknown protein structure (Rossmann & Blow, 1962).

The method relies on the fact that proteins sharing a high degree of sequence

homologies will have a similar structure. Therefore, it is very important that the


Camila Hernández

83

sequence identity between the phasing model and the target is high. By rule of

thumb, the amino acid sequences must be at least 25% identical and the r.m.s

deviation of the C-carbons less than 2 Å (Taylor, 2003).

In order to obtain the phase information, the search model must be correctly

oriented and positioned into the unit cell from the target crystal in such a way that

the resulting theoretical diffraction pattern is equivalent to the experimental

pattern. For this purpose, the molecular replacement process is divided into two

steps: a rotational search and a translational search. During these steps and for each

molecule, six parameters that describe how the search model is placed into the unit

cell of the target model are calculated: three rotational to indicate orientation and

three translational to indicate position. For both search and target models a

Patterson map is calculated. If the sequence identity shared by these models is high

enough, the Patterson maps will look similar and therefore correlate upon the

orientation and position of the molecules within the unit cell (Fujinaga & Read,

1987).

The Patterson maps are based on the Patterson function (Patterson, 1934), which is

similar to the electron density function (4.3.7). However, it uses the square of the

absolute value of the structure factor |Fhkl|2, thus the intensities that are measured

for every single reflection (4.3.7). Consequently, no phase information is required to

obtain the Patterson function:

The Patterson map is a vector map that contains information about the structure as

a sum of all interatomic distance vectors (intramolecular and intermolecular), which

indicate length and direction but not the location of the atoms. This fact is important

for the rotation function (RFn) (Rossmann & Blow, 1962) that relies on the precise

relative position of the atoms within the molecule indicated by the intramolecular

vectors, which depend only on molecular rotation.


Camila Hernández

84

The rotation function (RFn) is the product of the observed crystal Patterson

(Pobserved (u)) and the rotated model Patterson (Pmodel (R,u)) integrated over all

points u in the Patterson space within a sphere of radius rmax centered on the origin

and excluding the origin peak out to a radius rmin. The highest values for RFn are

obtained when the crystal Patterson and the rotated model Patterson coincide,

giving then the correct spatial orientation (Evans & McCoy, 2008).

Once the correct orientation of the model is obtained, the translational search by

means of a translation function estimates the correct position of the model in space.

The translation function correlates the observed intensities and the Patterson cross-

vectors of the symmetry-related molecules of the model upon movement in the unit

cell. When the correct position is found, the peak values of the translation function

correspond to the translation vectors between the symmetry-related molecules.

According to the RFn described above, which matches the intramolecular vectors in

the Patterson map, the translation function finds the best equivalent intermolecular

vectors dependent on the correct molecular position as well as the correct

orientation (Crowther & Blow, 1967).

There are different forms of translation function, the standard Patterson based T-

function (showed above) measures the similarity between the observed and

calculated Pattersons over the entire asymmetric unit. Another variant of this

function subtracts the known intramolecular vector component considering the

whole symmetry of the model. A conceptually simpler method compares the R-

factor and the correlation coefficient of Fobs and Fcalc at every point of the


Camila Hernández

85

translational search. In this case, the R-factor is used as a statistical measure to

compare the differences between the observed structure factor amplitudes |Fobs| of

the measured data, with the calculated structure factor amplitudes |Fcalc| for the

search model.

The purpose of this function is to minimize the difference between the structure

factor amplitudes thus decreasing the value of the R-factor. A lower value of the R-

factor indicates that a good solution was found; a perfect solution would give an R-

factor value of zero, however, experimentally a good solution generally gives R-

factor values around 0.3-0.4 (Rhodes, 2006).

Consequently, if the correct orientation and position of the search model are found,

the initial phases of the model can be calculated. This initial phase information

biased by the homologous structure model is then combined with the target

structure factor amplitudes obtained from the X-ray diffraction experiments in

order to calculate the electron density map.

4.3.9 Structure determination of Ks-Amt5

4.3.9.1 Cryo-cooling

It is commonly observed that protein crystals exhibit radiation damage when

exposed to X-rays leading to e.g. decay in the crystal diffraction. Radiation damage

occurs due to the production of water radicals that react with the protein destroying

the crystal lattice. In order to avoid this, protein crystals are usually flash-frozen in

liquid nitrogen and then exposed to X-rays by cooling them at 100 K with a constant

flush of nitrogen gas during the measurement. However, the process of freezing can

lead to the formation of ice crystals that can mask the diffraction pattern of the

protein by the presence of ice rings. Thus, it is important to find a cryoprotectant


Camila Hernández

86

that preserves the physical state of the protein crystal, prevents the formation of ice

crystals and also ensures optimal data quality (Garman & Owen, 2006).

For Ks-Amt5 crystals, PEG 400 with a final concentration of 20% (v/v) was

successfully used as cryoprotectant.

4.3.9.2 Data collection and processing

Crystal diffraction was tested in-house at CuK-radiation of =1.5418 Å, using a

rotating copper anode (Micromax 007 HF, Rigaku, Tokyo, Japan) with a Saturn 944+

CCD-detector (Rigaku, Tokyo, Japan) or with a Mar345 image plate (Marresearch,

Norderstedt, Germany). In order to obtain higher resolution data, well-diffracting

crystals were stored and used for further data collection at a synchrotron.

iffraction data were collected with rotation angles of 0.5˚-1˚ per image for 360˚

using X-rays with 1.0 Å wavelength. Data was collected at the X06SA and X06DA

beamlines from the Swiss Light Source (Paul Scherrer Institute, Villingen,

Switzerland) using a Pilatus 6M or a MX-225 CCD detector, respectively.

The collected data sets were indexed and integrated using iMOSFLM (Leslie, 1992).

Additional symmetry elements were determined with POINTLESS (Evans, 2006).

Integrated data was scaled using SCALA (Evans, 2006) from the CCP4 software suite

(Collaborative Computational Project Number 4, 1994).

4.3.9.3 Structure solution

The structure of Ks-Amt5 was solved by molecular replacement. MR was performed

using the MOLREP program (Vangin & Teplyakov, 1997) of the CCP4 software suite

(Collaborative Computational Project Number 4, 1994). For the initial data set, the

initial models used were chosen according to sequence similarity: the Af-Amt1

protein model of A. fulgidus (Andrade et al., 2005) sharing a 34% sequence identity

with Ks-Amt5 for the membrane domain and the cytoplasmatic portion of the sensor

histidine kinase from T. maritima (Marina et al., 2005) (32% sequence identity) for


Camila Hernández

87

the cytoplasmatic domain. Both models were obtained from the protein data bank

under the PDB accession codes 2B2H and 2C2A, respectively.

The resulting refined structure of Ks-Amt5 was later used as model for further

molecular replacement in data sets at higher resolution.

4.3.9.4 Model building and refinement

The Ks-Amt5 structural model was built into the electron density map obtained after

rigid-body refinement with REFMAC5 (CCP4 software suite; Murshudov et al., 1997)

using the program COOT (Emsley & Cowtan, 2004; Emsley et al., 2010). Further, the

structural model was improved in cycles of alternate building and restrained

refinement with REFMAC5. The final model was validated with the program

PROCHECK (Laskowski et al., 1993).

4.4 Graphical representations

Illustrations of the protein structures were made using PyMOL (DeLano, 2002;

Schödinger LLC, 2009). Electrostatic surface potentials were calculated using

DELPHI (Honig and Nicholls, 1995; Rocchia et al., 2001) assuming standard charges

for amino acids. Sequence alignments were carried out with CLUSTALW (Thompson

et al., 2002) and plotted with CLC sequence viewer (CLC bio, 2005).

Results and discussion

Camila Hernández

88

5 Results and discussion

The increase in available genome sequences has facilitated the study of interesting

proteins involved in relevant processes in cells. Searches for amt sequences show

interesting and totally undescribed proteins with extramembraneous domains with

variable functions. In this work one of these proteins (Ks-Amt5) was chosen from

“Ca. Kuenenia stuttgartiensis”, an anammox bacteria member of an ecologically and

environmentally important group of microorganisms, which play a crucial role in

the removal of undesired ammonium from municipal and industrial waste water.

5.1 Sequence analysis of Ks-Amt5

Sequence analyses were carried out in order to gain insight into the amino acid

composition and to identify conserved residues and motifs described for Amt

proteins and histidine kinases. The sequence alignment of Ks-Amt5 was performed

with ClustalW2 (Larkin et al., 2007) and includes the Af-Amt1 and Ec-AmtB proteins

with known crystal structures and five other members of the Amt/Rh family chosen

from a BLAST search. Topology predictions were carried out with TMHMM

(Sonnhammer et al., 1998; Krogh et al., 2001) in order to identify secondary

structure features for the Amt domain of Ks-Amt5. Based on these results, the Amt

domain (residues M1-D412) of Ks-Amt5 presents eleven transmembrane helices

(Figure 21). In contrast to Ec-AmtB, the Ks-Amt5 sequence does not contain a

cleavable signal peptide and consequently the N-terminus of the protein forms part

of the first transmembrane helix that crosses the membrane. The conserved amino

acids presumably involved in the translocation of ammonium are located in

transmembrane helices 3, 4, 5, 6 and 10 of Ks-Amt5. In the predicted recruitment

site W144 (W137 in Af-Amt1) and S227 (S208) residues are present, as well as the


Camila Hernández

89

F103 and F223 (F96 and F204) for the phenylalanine gate and the twin-his pair

H171 and H326 (H157 and H305) (Andrade et al., 2005) (Figure 21).

Figure 21: Multiple sequence alignment of the transmembrane domain of Ks-Amt5 with

Amt/Rh proteins. Ks: “Ca. Kuenenia stuttgartiensis”; Af: Archaeoglobus fulgidus; Ec: Echerichia coli;

At: Arabidopsis thaliana; Le: Lycopersicum esculentum; Ne: Nitrosomonas europaea. Transmembrane

helices, shown in blue (TM1-TM11), are indicated according to the Ks-Amt5 structure. Conserved

residues are shown in black. The orange boxes depict the highly conserved amino acids supposed to

be involved in the ammonium transport.


Camila Hernández

90

The C-terminal domain (Ks-Kin) of Ks-Amt5, regarding residues F413 to K679,

exhibits a high similarity to various histidine kinases from the two-component

signal transduction pathway (Figure 13). In particular, it shares 32 % sequence

homology with the cytoplasmatic portion of a sensor histidine kinase with known

crystal structure, TM083 from the thermophile Thermotoga maritima, previously

described in section (3.4.3). The alignment result is clear with regards to the

presence of all characteristic motifs, H, N, F and G boxes in the DHp and CA domains

of histidine kinases and indicates H460 as the phosphorylation site.

In addition, secondary structure predictions were carried out using PSIPRED (Jones,

1999; Bryson et al., 2005) and the predicted topology was compared with the

secondary structure elements found for the TM083 structure. The results indicate

the presence of the conserved structural features for the DHp domain (alpha helix 1

from R415 to I480 and alpha helix 2 from K486 to E518) and CA domain (five -

strands and three -helices)(Figure 22).

Figure 22: Multiple sequence alignment of Ks-Amt5 cytoplasmic domain with other histidine

kinases. Ks: “Ca. Kuenenia stuttgartiensis”; TM: Thermotoga maritima; Ty: Thermodesulfovibrio

yellowstonii; Mb: Methanoccocoides burtonii; Mm: Methanosarcina mazei. Conserved amino acid

residues are shown in black. Secondary topology predictions for Ks-Amt5 are shown in orange (-

helices) and green (-sheets). The histidine kinase characteristic motifs are marked in blue boxes and

the phosphorylation site (H469) is highlighted in blue. The sequence alignment was truncated

according to the sequence of Ks-Amt5.


Camila Hernández

91

Interestingly, Ks-Amt5 is a previously undescribed and entirely new member of the

Amt family containing a histidine kinase extra-membrane domain with about 30

kDa (residues F413-K679) and a typical Amt transmembrane domain with eleven

transmembrane helices (residues M1-A408). From the sequence analysis and

topology predictions it is evident that the structure of the Amt domain of Ks-Amt5

resembles the structure of other Amt proteins like Ec-AmtB and Af-Amt1. Due to the

presence of the conserved residues W144 and S227 suggested to form the

recruitment site for ammonium as well as other residues involved in the transport

mechanism, it seems likely that Ks-Amt5 can bind and translocate ammonium across

the membrane besides the histidine kinase activity as a signal-transduction protein.

5.2 Cloning and mutagenesis of Ks-Amt5

All constructs used in this work were cloned via NdeI and XhoI restriction sites into

pET21a and pET28a or pET15dT vectors. To produce the full-length protein (Ks-

Amt5) containing both the Amt domain (Amtdom) and the histidine kinase domain,

the complete amt5 gene (2036 bp) was cloned into the pET21a vector. Shorter

constructs were also designed for the production of the histidine kinase protein

(residues F413-K679) in the absence of the integral membrane domain and vice

versa (Amtdom, residues M1-D412). For this, the amt5 gene was truncated in the

region encoding for the amino acids D412-F413 using PCR with appropriate primers

and inserted into the respective plasmids (pET21a, pET28a, pET15dT).

A shorter version of the Ks-Kin construct (Ks-KinS) was also designed. For this

purpose, 26 amino acids at the N-terminal part of the protein were cut resulting in

the construct M439-K679 (Figure 23). Using site-directed mutagenesis a new NdeI

restriction site was inserted into the pET28a::kin at a position that encodes for

M439. Upon restriction digestion with NdeI and XhoI the truncated version of the kin

gene was subsequently isolated and ligated into the pET21a vector.


Camila Hernández

92

For functional studies, variants of Ks-Kin were designed and obtained by site-

directed mutagenesis experiments in the original constructs. For pET15dT::kin, the

histidine residue H460 identified as the phosphorylation site was mutated to a non-

polar amino acid, alanine, in order to create a variant that would function as a non-

functional blank in phosphorylation experiments. Another variant for pET15dT::kin

resulted from a double mutation (I612W/S66W) in the ATP binding pocket and was

designed to sterically prevent ATP binding.

Figure 23: Constructs used for protein production. For the variants of Ks-Kin the site-directed

mutagenesis experiments were carried out using the original plasmids. The amino acids considered

for mutations are indicated as H460 (phosphorylation site) and I612/S666 (ATP-binding site).

All constructs with the exception of pET21a::amtdom were successfully expressed

and purified.

5.3 Protein production

Ks-Amt5 and Ks-Kin proteins holding a C-terminal His6-tag, both N- and C-terminal

His6-tag or a N-terminal His10-tag could be produced in E. coli C43 (DE3) or E. coli


Camila Hernández

93

BL21 (DE3) cells. Test expressions with different media (LB, TB, auto-inducing,

minimal media) and temperatures (18, 20, 30, 37 ˚C) were performed in order to

find the best expression conditions for all constructs. Subsequently, western blots of

whole cell fractions were carried out after harvesting the cells and resuspending

them in water with 20 µl of water added per 0.1 Au of OD600. In the case of Ks-Amt5,

the protein was overproduced by cold induction with 0.4 mM IPTG after which

grown continued at 20 ˚C for 18 hours. The overproduction of Ks-Kin, Ks-KinS and

variants was generally high, resulting in high protein yields. Generally cultures were

grown at 30 ˚C and gene expression was induced with 0.4 mM IPTG. For all protein

productions, LB medium was chosen for large-scale expression.

Figure 24: Ks-Amt5 and Ks-Kin production detected in western blots. Results of experiments

showing the expression of pET21a::ks-amt5 and pET15dT::ks-kin in E. coli C43 (DE3) and BL21 (DE3)

cells respectively. Sample T0 was taken right after induction with IPTG, T1 after 1 hour (for Ks-Amt5,

18 hours) and T2 after 2 hours of induction. The PageRuler Plus Prestained Protein Ladder

(Fermentas) was used as molecular weight marker (MW).

After optimizing the expression conditions for the constructs, western blots were

also performed to track the level of production of the His-tagged proteins (Figure

24). Generally, samples were collected at the time of induction (T0), one hour after


Camila Hernández

94

induction (T1), two hours after induction (T2) and for Ks-Amt5 after 18 hours. The

cultures reached the beginning of the growth stationary phase at about T1 for Ks-

Amt5 and T2 for Ks-Kin and its variants.

The expression levels under the conditions mentioned yielded sufficient amounts of

protein, which was further used in crystallization trials and functional assays.

5.4 Protein purification

Protein purification followed established methods for the Amt homolog, Af-Amt1

(Andrade et al., 2005b) with modifications in buffer compositions (4.2.3.3 and

4.2.3.4). As an improvement for the protocol an additional 40 mM imidazole pH 8.0

was added to the loading buffer for IMAC.

5.4.1 Ks-Amt5

The purification of Ks-Amt5 did not show a significant dependence on the

detergents used. Ks-Amt5 was successfully purified using a variety of detergents

including non-ionic detergents such as maltosides (D9M to D13M) and OGP and a

zwitterionic detergent, LDAO. In addition to dilutions of a single detergent type,

D9M, D10M, D11M, DDM, D13M, LDAO and OGP, 1:1 mixtures of two different

detergents were tested (concentrations are shown in Table 8). All of the screened

detergent solutions were able to stabilize the protein and resulted in monodisperse

samples. Based on previously solubilization tests, DDM was identified as the best

detergent for the solubilization of Ks-Amt5. Consequently, the solubilization of

membranes was performed with DDM at a final concentration of 1 %. Since all

purifications of Ks-Amt5 were very similar, only the purification with a mixture of

0.65 % D9M plus 0.03 % DDM is discussed.

The solubilized E. coli C43 (DE3) membranes containing Ks-Amt5 were purified via

IMAC as described previously (4.2.3.3). Affinity chromatography (Figure 25A and


Camila Hernández

95

25B) yielded highly pure Ks-Amt5 (Figure 25D). The fractions obtained and

containing Ks-Amt5 protein were pooled and concentrated to 300-500 µl by

ultrafiltration using a 50 kDa MWCO concentrator. The concentrated protein was

further purified by SEC (4.2.3.4) to obtain a homogenous and monodisperse sample.

The SEC profile obtained from a Superdex S200 10/300 column showed a high

elution peak for the Ks-Amt5 with a smaller peak that presented minimal

absorbance at the exclusion volume probably due to oligomeric aggregates (Figure

25C). The retention volume of the Ks-Amt5 peak (VEl = 10 ml) corresponds to a

molecular size of approximately 282 kDa according to the calibration curve, log

(MW) = - 0.2055 * VEl + 4.5054, R2 = 0.982. The theoretical molecular weight of a

single Ks-Amt5 monomer including the His6-tag is about 75.2 kDa. Ks-Amt5 is

expected to form a trimer and furthermore to exhibit a higher molecular weight due

to the formation of a protein-detergent complex with the detergent molecules

present in the purification buffers (aggregation numbers of D9M and DDM are ~ 25

and ~78-149 respectively; VanAken et al., 1986). Therefore, the 282 kDa mass can

be interpreted as the molecular weight for the trimeric form of Ks-Amt5 (75.2 x 3=

225.6 kDa) including detergent molecules (± 56.4 kDa).

Subsequently, the Ks-Amt5 protein was further concentrated to ~7-10 mg/ml

(determined by a BCA test, 4.2.3.5) by ultrafiltration using a 100 kDa MWCO

concentrator. Generally, the yields obtained were about 0.1-0.3 mg protein from 1 L

culture. Crystallization trials were immediately performed with freshly purified

protein. For long-term storage the protein was flash frozen in liquid nitrogen and

stored at -80 ˚C.

In addition to SEC, blue-native PAGE experiments were carried out to determine the

oligomeric states of the Ks-Amt5 protein. BN-PAGE experiments (4.2.3.10) were

carried out using purified protein after size exclusion chromatography. The results

of BN-PAGE confirmed the assumption that the Ks-Amt5 is a trimer in solution

(Figure 25E). One distinct band was observed at 232 kDa corresponding to the

already estimated MW for the trimeric form of Ks-Amt5. In addition, higher


Camila Hernández

96

oligomeric aggregates, hexamer and dodecamer, were detected. This might explain

the presence of the low intensity exclusion volume peak obtained in the SEC profile.

Figure 25: Purification of Ks-Amt5 in a 1:1 mixture of 0.65 % D9M + 0.03 % DDM. A. Ks-Amt5

IMAC purification showing the overall chromatogram of the His-trap run. B. Detail of the two elution

steps (5 % and 50 %) represented by the imidazole gradient (green line). C. Ks-Amt5 SEC

chromatogram. The Ks-Amt5 peak is labeled with the corresponding retention volume. D. SDS-PAGE

analysis of pure Ks-Amt5. Lanes include the unstained protein marker (MW) (Fermentas) and the

protein samples after affinity (HT) and size exclusion chromatography (GF). The Ks-Amt5 monomer

migrates with an apparent molecular mass of about 60 kDa, smaller than its calculated molecular

weight of 75.2 kDa. These differences in SDS-PAGE band migration are characteristic for membrane

proteins. E. BN-PAGE results indicate that Ks-Amt5 is a trimer in solution. The visible bands can be

designated to different oligomerization states (dodecamer, hexamer and trimer). The protein native

marker is indicated as MW (HMW Native marker, GE Healthcare).


Camila Hernández

97

5.4.2 Ks-Kin and variants

Since the Ks-Kin is a soluble cytosolic protein, detergents were not needed for

protein purification. Despite the difference in length as well as the position of the

affinity tag in the kin constructs, all purifications of Ks-Kin and variants closely

resemble each other in terms of their experimental execution and profiles. Thus,

only the purification of the Ks-Kin (pET28a::kin construct) is shown and discussed

exemplarily.

The cytosolic fraction, containing overproduced Ks-Kin, was obtained by

centrifugation of the disrupted E. coli BL21 (DE3) cells and purified via IMAC

(4.2.3.3). High amounts of pure protein were obtained after affinity chromatography

(Figures 26A, 26B and 26D). The fractions containing Ks-Kin protein were pooled

and concentrated to 500-1000 µl by ultrafiltration using a 10 kDa MWCO

concentrator. The concentrated protein was further submitted to a size exclusion

chromatography to evaluate the homogeneity the sample. SEC was carried out using

a Superdex S200 10/300 or S200 26/60 according to the amount of protein to be

injected and following the protocol as previously described (4.2.3.4). Generally, a

single symmetric peak was observed (Figure 26C) indicating a homogeneous

protein sample with no apparent sign of aggregation. After SEC, the Ks-Kin protein

was concentrated to ~10-30 mg/ml (determined by a BCA test, 4.2.3.5) by

ultrafiltration using a 30 kDa MWCO concentrator. High yields of pure protein were

obtained (Figure 26C) in amounts of 3-6 mg from 1 L culture. Crystallization trials

were performed directly afterwards with freshly purified protein. For long-term

storage, the protein was aliquoted and flash frozen in liquid nitrogen and stored at -

80 ˚C.


Camila Hernández

98

The molecular weight of Ks-Kin was deduced from the SEC results. The protein

eluted at a volume of 196.95 ml from a Superdex S200 26/60 column (Figure 26B),

which corresponds to a molecular weight of 61.9 kDa according to the calibration

curve, log (MW) = - 0.0155 * VE + 7.8445, R2 = 0.985.

The theoretical molecular weight of a single Ks-Kin monomer including the

purification tags (N and C terminal His6-tag, in this example) is 31.7 kDa.

Accordingly, the calculated molecular weight of 61.9 kDa corresponds to a Ks-Kin

dimer. In addition, BN-PAGE analyses (4.2.3.10) were performed to corroborate the

oligomerization state of Ks-Kin. Although it was difficult to obtain a good native gel

for the cytosolic domain, it was possible to visualize a protein band at about 62 kDa,

which indicates the presence of a dimeric form of Ks-Kin in solution. This result

agrees with experimental data obtained for other histidine kinases such as Tm-

CheA, and TM083 (Bilwes et al., 1999; Marina et al., 2005) where SEC results

indicate the presence of a protein dimer. The difference in the oligomerization

behavior of the full-length protein (Ks-Amt5) and the cytosolic domain (Ks-Kin)

further poses the question of how the cytosolic domain is organized in the full-

length protein and moreover, how it might function. We aim to answer these

questions by solving the molecular structure of Ks-Amt5 by means of X-ray

crystallography in various functional states.


Camila Hernández

99

Figure 26: Purification of Ks-Kin. A. Ks-Kin IMAC purification showing the overall chromatogram of

the His-trap run. B. Detail of the two elution steps (5 % and 50 %) represented by the imidazole

gradient (green line). C. Ks-Kin SEC chromatogram. The Ks-Kin peak is labeled with the

corresponding retention volume. D. SDS-PAGE analysis of pure Ks-Kin. The Ks-Kin monomer

migrates with an apparent molecular mass of about 31 kDa. A higher weaker band at about 64 kDa

corresponds to the dimer. Lanes include the unstained protein marker (MW) (Fermentas) and the

protein samples after affinity (HT) and size exclusion chromatography (GF). E. BN-PAGE results

indicate that Ks-Kin is a dimer in solution. The visible bands can be designated to different

oligomerization states (hexamer, tetramer and dimer). The protein native marker is indicated as MW

(HMW Native marker, GE Healthcare).


Camila Hernández

100

5.5 Crystallization of Ks-Amt5

Although a significant leap forward could be observed in the last couple of years

based on the number of new membrane protein structures published in the Protein

Data Bank, the determination of membrane protein structures by X-ray

crystallography still presents a challenge and confronts the experimenter with many

difficulties (Lacapère et al., 2007). As membrane proteins are surrounded by lipids,

they have to be extracted from the lipid environment in order to purify them. For

this, specific detergents are required. Consequently, the optimal choice of detergents

used for the extraction and purification of membrane proteins is very important

since they must maintain the native folding of the protein and stabilize it without

compromising its functional state (Newby et al., 2009). Generally, a longer chain

detergent works best to extract the protein from the membrane. As a consequence,

these detergents may recover higher amounts of the membrane protein from the

lipid bilayer. On the other hand, shorter chain detergents are better suited to

facilitate protein-protein contacts and thus crystallization. Moreover, the use of

detergent mixtures might be helpful for the crystallization of membrane proteins. A

mixed detergent micelle containing multiple detergents is potentially able to

significantly ameliorate protein stabilization and crystallization. This was

successfully demonstrated for G protein-coupled receptors and bacteriorhodopsin

where protein purified in bicelles, composed of two detergents and/or lipids, was

used for the final structure determination (Faham et al., 2002; Chiu et al., 2008).

In the case of Ks-Amt5, crystals could only be obtained after purification based on a

1:1 detergent mixture (D9M+DDM, D10M+DDM, DDM+D13M or D10M+LDAO)

(concentrations used are shown in Table 8). Initial crystals appeared after 10-15

days in an Index crystallization screen condition (Hampton research, Aliso Viejo,

USA) composed of 25 % PEG 3350 and 0.1 M HEPES buffer pH 7.5 and stored at 20

˚C (Figures 27A and 27B). Under this condition the original crystals could be

reproduced with a suitable size for X-rays experiments. Crystals were further

obtained in a variety of PEG 3350 concentrations (25-32 %) and HEPES buffer pH


Camila Hernández

101

7.5 concentrations (0.1-0.15 M) (Figures 27C and 27D). Under other conditions of

the Index screen such as 30 % PEG 2000 plus 0.1 M potassium thiocyanate (Figure

27E) and 30 % PEG 2000 plus 0.15 M potassium bromide (Figure 27F) single

crystals appeared, although in these cases crystals were not reproducible.

Additional crystallization trials using detergent screens, additive screens (Hampton

research) and microseeding were performed in order to optimize and improve the

crystal diffraction quality. Conditions including different additives led to the

formation of crystals, however, the best crystals were obtained in finescreens (4.3.3)

by simply changing the buffer type and pH and slightly reducing the PEG

concentration. The crystallization condition which produced generally well-

diffracting crystals of Ks-Amt5 purified in 0.65 % D9M + 0.03 % DDM, was 24 % PEG

3350 and 0.1 M MES pH 7.0 (Figures 27G and 27H). Crystals presented a hexagonal

prism shape and in some cases with a pointy edge. The crystal form was similar in

all crystallization trials and only variations in width and length were observed.

In addition, crystals of Ks-Amt5, purified in 0.65 % D9M + 0.03 % DDM and obtained

in 24 % PEG 3350 + 0.1 M MES pH 7.0, were further used for soaking experiments.

For this, a mixture containing the corresponding reservoir and an ATP or non-

hydrolyzable ATP solution (AppCp, ATPS or ATPS) to a final concentration of

1 mM was prepared. Crystals were soaked in this solution for 1-5 min aiming for the

introduction of an ATP molecule to the binding pocked of the histidine kinase

domain. By means of these experiments it was expected to observe different

conformations of the protein after structure determination.


Camila Hernández

102

Figure 27: Ks-Amt5 crystals. A and B. Initial hit for Ks-Amt5 crystals with protein purified in 0.2 %

D10M + 0.03 % DDM and 0.65 % D9M + 0.03 % DDM respectively. C and D. Crystals of Ks-Amt5 in

different concentrations of PEG 3350, 26 % and 32 %, with protein purified in 0.65 % D9M+ 0.03 %

DDM. E and F. Non-reproducible crystals of Ks-Amt5 in 30 % PEG 2000 plus potassium thiocyanate

and potassium bromide, respectively. The protein was purified in 0.2 % D10M+ 0.03 % DDM. G and H.

Best crystallization condition for Ks-Amt5 with protein purified in 0.65 % D9M + 0.03 % DDM.

Crystals obtained diffracted up to 2.1 Å.

5.6 Crystallization of Ks-Kin

For the crystallization of the cytosolic domain of Ks-Amt5 all different constructs of

Ks-Kin were used, in all cases with the affinity tag present (Figure 23).

Crystallization trials included a variety of initial crystallization screens (Footprint,

Index screen, Natrix screen, Crystal screen, Morpheus screen, JB screens and PEG

Grid screens) and a variety of temperatures 4 ˚C, 20 ˚C, 25 ˚C, 30 ˚C and 37 ˚C for the

storage of the crystal plates. However, no protein crystals appeared. As mentioned

before, the cytosolic domain presents a DHp plus a CA domain which contains an

ATP binding site. Aims to achieve a higher homogeneous sample, that is in only one

conformational state, co-crystallization trials with ATP and non-hydrolysable ATP

analogues (AppCp, ATPS, ATPS) were performed using different concentrations


Camila Hernández

103

(0.1 mM – 2mM) of this as additive, either in the drop or pre-incubated (for 30 min –

1 h) with the protein solution containing 5-10 mg/ml protein, 20 mM Tris-HCl, 100

mM NaCl and 1 mM MgCl2. Despite this, no crystals were obtained. In continuation

of these efforts, a second construct with a shorter α1-helix, Ks-KinS (residues

M439-K679) was designed in order to truncate a possible flexible region that might

interfere with the crystallization process. With this construct, however

crystallization screens and conditions, as well as co-crystallization trials with ATP

and non-hydrolysable ATP analogues did not lead to the growth of crystals.

Parallel experiments using the Ks-Kin construct (pET28a::kin) were carried out with

another structural technique to determine the overall structure of this domain

(5.11).

5.7 Data collection and processing

Initial crystals of Ks-Amt5 diffracted only to a maximum resolution of 11-8 Å and

usually showed an anisotropic diffraction pattern. In modern X-ray crystallography,

the choice of cryoprotectants is crucial for the collection of good quality data. High

molecular PEGs have been proven to be good cryoprotectants (Berejnov et al.,

2006). However, they have to be in a suitable concentration generally above 25 % to

avoid the formation of water crystals that interfere (superimpose) with the X-ray

diffraction of the protein crystal. Therefore, before mounting the crystals in cryo

loops (Hampton research, Aliso Viejo, USA), the crystallization drop was exposed to

air for 5-10 min to slightly increase the original PEG 3350 concentration (24 %) in

the drop through the evaporation of water. Contrary to the use of 24 % PEG 3350 as

cryoprotectant, this procedure improved the diffraction limit of the Ks-Amt5

crystals from 8 Å to 3.5 Å. Consequently, this allowed the collection of the first

diffraction data set of Ks-Amt5 crystals at 3.5 Å resolution. Additionally,

commercially available additive and detergent screens (Hampton research, Aliso

Viejo, USA) were used in order to improve the crystal diffraction quality.


Camila Hernández

104

Furthermore, other cryoprotectant solutions, such as 10 % (v/v) glycerol and

10 % (v/v) (R,R)-2,3-Butanediol were tested. However, these cryoprotectants did

not show an improvement in thecrystal diffraction. For Ks-Amt5 crystals the best

cryoprotectant turned out to be PEG 400. The used of PEG 400 as cryoprotectant

was earlier described to improve the lattice order and stability of crystals (Xiao &

Gamblin, 1996). In practice, a small amount of the reservoir solution was mixed with

PEG 400 at a final concentration of 20 %. Subsequently, Ks-Amt5 crystals were

soaked in this mixture (1-2 min) before freezing in liquid nitrogen. As a result, the

diffraction limit was further improved from 3.5 Å to 2.1 Å. At this so far maximum

resolution (2.1 Å), a data set was collected at the X06SA beamline at the Swiss Light

Source (Villingen, Switzerland) at 100 K. The diffraction data was processed and

analyzed as previously described (4.3.9.2). Like Ec-AmtB crystals (Khademi et al.,

2004; Zheng et al., 2004), the space group of the Ks-Amt5 crystals was determined

to be P63 with cell parameters a = b = 99.77 Å, c = 89.08 Å, = = 90˚, = 120˚ and a

Mathews coefficient of VM = 2.68 Å3/Da that corresponds to a 53.71 % solvent

content (Matthews, 1968; Matthews, 1976). The details of data collection and

processing statistics are summarized in Table 20.

5.8 Overall structure and crystal packing

The first crystal structure of Ks-Amt5 was solved at 3.5 Å by molecular replacement

with the Af-Amt1 structure (PDB code: 2B2H) as a search model. Afterwards, the

structure of Ks-Amt5 at higher resolution (2.1 Å) was solved as well by MR with the

3.5 Å Ks-Amt5 structure as the search model. In X-ray protein crystallography the

resolution limits are important, while the lower resolution diffraction spots allow

the determination of the overall structure, the higher resolution spots enable the

visualization of details that are generally crucial to the description of the

functionality of the protein. The structural model of Ks-Amt5 that will be further

discussed is based on the diffraction data set at a maximum resolution of 2.1 Å.


Camila Hernández

105

Table 20: X-ray data processing and refinement statistics for Ks-Amt5. Data set recorded at X06SA at the Swiss Light Source PSI, Villingen, Switzerland). Values in parentheses indicate the

highest resolution shell.

Data processing and refinement statistics for Ks-Amt5 Wavelength (Å) 1.000 Space group P63 Unit cell parameters a,b,c (Å); ,, (˚) 99.772, 99.772, 89.084; 90, 90, 120 Resolution limit (Å) 2.1 Resolution range (Å) 62.02-2.1 Number of reflections, unique 29455 (4272) Completeness overall (%) 99.84 (99.7) Multiplicity (%) 10.2 (10.1) Rmerge overall

1 9.1 (70.8) Rpim

2 3 (23.2)

Rvalue overall3(%) 16.07

Rvalue free 4(%) 20.09

Mean I/Sig (I) 21.5 (3.7) Cruickshank’s PI (Å)5 0.1681 R.m.s. deviations from ideal values r.m.s.d bond lengths (Å) 0.0194 r.m.s.d bond angles (˚) 2.1163

Average B values (Å2)

Protein main chain atoms 32.490 Protein all atoms 34.594

, angle distribution for residues6

In favored regions (%) 92.3 In allowed regions (%) 4.3 In outlier regions (%) 3.4 1 Rmerge = hkl [(i |Ii - <I>|)/ iIj] 2 Rpim = hkl N/N-1 [(i |Ii - <I>|)/ iIj] (Weiss et al., 2001) 3 Rvalue = hkl ||Fobs| - |Fcalc|| / hkl|Fobs| 4 Rfree is the cross-validation R factor computed for the test set of 5 % of unique reflections (Brünger, 1993) 5 DPI: Diffraction precision indicator (Cruickshank, 1999) 6 Ramachandran (Ramachandran & Sasisekharan, 1968) statistics as defined by PROCHECK (Laskowski et al., 1993)

As mentioned before, in all cases, crystals belonged to the hexagonal space group

P63 with one Ks-Amt5 monomer per asymmetric unit. The crystal packing is

determined by the side contacts between the periplasmatic and cytoplasmatic loops

of the integral membrane domains (Figure 28). Consequently, each Amt monomer

forms crystal contacts through interactions between residues of loops 2, 5, 7 and 10.

The symmetrically related Amt monomers form trimers that are oriented in a three-

fold axis with the N-terminal regions facing the C-terminal regions of a neighbor

trimer (Figure 28A). Each monomer extends ~ 52.8 Å parallel to the three-fold axis


Camila Hernández

106

and the trimer has an overall diameter of ~ 82 Å in the plane of the membrane

(Figure 28B).

Figure 28: Crystal packing of Ks-Amt5. Each asymmetric unit contains a Ks-Amt5 trimer. A. Overall

view of the P63 crystal packing. The trimers are intercalated with the N-terminal side facing the C-

terminal side of the neighbor trimers. The empty region below the Amt domain was expected to

reveal the position for the histidine kinase domain. The crystal contacts are indicated with a circle. B.

View of the three-fold symmetry of the trimer.

Below each trimer molecule there is an empty space, which corresponds to the

position of the cytosolic domain not visible in the structure (Figure 28A). During the

molecular replacement search it was not possible to localize the homologous

structural model TM083 (PDB code: 2C2A, Marina et al., 2005) of the Ks-Kin.

Therefore, MR searches were carried out with truncated versions of the original

model (model A residues E240-R317 and model B residues E325-R480) in order to

localize particular domains of the histidine kinase protein such as the catalytic and

the dimerization domains. Despite the different search model, the high-resolution


Camila Hernández

107

data (2.1 Å) and the data completeness (99.84 %), the histidine kinase domain was

not visible in any of the data sets analyzed. Some electron density was observed in

the C-terminal part of each monomer; even so, it was not sufficient to reconstruct

the histidine kinase domain of the Ks-Amt5.

5.9 Ks-Amt5 monomer

As predicted, the Ks-Amt5 monomer consists of residues M1-A408 forming eleven

transmembrane helices, TM1-11 (Figure 29A). As seen in other Amt proteins, the

monomer of Ks-Amt5 presents an internal pseudo two-fold symmetry with an axis

in the membrane plane. It consists of two halves comprised of TM1-TM5 on one side

and TM6-TM10 on the other. As is the case with Ec-AmtB and Af-Amt1, the two

pseudo-symmetric halves of the Amt5 monomer are held and stabilized by the

remaining transmembrane helix TM11 (Figure 29B).

For each momomer, three detergent molecules of DDM were identified on the

protein membrane surface between TM2-TM4, TM5-TM10 and TM4-TM11. Its

hydrophobic tails appear to surround the transmembrane helices while the

hydrophilic head groups point to the cytoplasm or to the periplasm respectively

(data not shown). The location of the detergent molecules in the structure of Ks-

Amt5 gives an additional indication of the position occupied by lipid molecules in

the real cell membrane.

B-factor analysis of the Ks-Amt5 model was performed to identify flexible regions.

The B-factor values are indicators of the attenuation in the X-ray scattering due to

thermal motion of the atoms (4.3.7). Thus, low B-factors indicate well-ordered parts

in the structure and high B-factors represent flexible portions. Overall, the Amt

transmembrane domain of Ks-Amt5 is well-ordered with average low B-factors in

the range of 10-40 Å (Figure 29C). Moderate flexibility was observed for the loops

involved in the crystal packing (loops, 2, 5, 7 and 10) with slightly higher B-factor

values (50-70 Å). On the other hand, the C-terminal extension composed by amino


Camila Hernández

108

acids E403 to A408, shows high B-factor values (up to 112 Å) (Figure 29D). The

flexibility of this region might explain the lack of a structural model for the

cytoplasmatic domain in Ks-Amt5.

In the current Ks-Amt5 model, the extramembrane HK domain at the C-terminal

part of the Amt domain appears to be positioned in close proximity to loop 5 and

blocks the cytoplasmic exit channel of the neighbor monomer (Figure 29D) . This

loop region in other Amt proteins such as Ec-AmtB, is involved in the interaction

with nitrogen regulatory proteins like GlnK (Conroy et al., 2007). Andrade et al.,

2005, suggested that movements in this loop region, which showed elevated B-

factor values for Af-Amt1, could permit conformational changes during transport if

they occur. In Ks-Amt5 loop 5 is rather ordered in the structure. However, an

interaction with the C-terminal extension of the neighboring monomer might

indicate cooperativity with regard to the histidine kinase domain activity.

Nonetheless, as of yet this is a hyphothesis, as the structure of Ks-Amt5 lacks the

complete cytoplasmatic domain and the C-terminal extension is highly flexible.


Camila Hernández

109

Figure 29: Structural details of Ks-Amt5 and B-factors analysis. A.The Ks-Amt5 monomer is

rainbow-colored with the N-terminus in blue and the C-terminus in red. The transmembrane (TM)

helices are indicated. B. 90 ˚C top view of A showing the pseudo two-fold symmertry of the Ks-Amt5

monomer. The two halves formed by TM1-TM5 and TM6-TM10 are indicated. C. B-factor putty

representation of the Ks-Amt5 monomer. Warm colors indicate high B-factor values. Loops 2, 7 and

10, which are involved in the crystal packing, present a moderate flexibility. The higher B-factor

values were obtained for the C-terminal region. D. C-terminal view of the Ks-Amt5 trimer in B-factor

putty representation. The flexibility of the C-terminus could be a reason for the disorder of the

histidine kinase domain, which is not visible in the structure.


Camila Hernández

110

Calculations of the Ks-Amt5 electrostatic surface potentials showed neutral

electrostatic potentials within the membrane plane, which could be interpreted as

an indication of the hydrophobic regions of the protein (Figure 30). The periplasmic

and cytoplasmic side of the Ks-Amt5 trimer present regions of negative and positive

electrostatic potentials, respectively, which agrees with the positive-inside rule for

membrane proteins (von Heijne & Gavel, 1988). Interestingly, although the

periplasmic entrance of the ammonium channel does not present a significant

charge, the cytoplasmic exit of the channel is slightly negative, suggesting the

transport of the charged species (NH4+) in Ks-Amt5.

Figure 30: Trimeric structure of Ks-Amt5 and electrostatic surface potentials. From left to right,

different views on the trimeric Ks-Amt5 model as seen from the N-terminal side, membrane plane

and C-terminal side, respectively. Top: representation of the electrostatic surface potential. Bottom:

carton representation.


Camila Hernández

111

In order to obtain a structural model for the histidine kinase domain, additional

experiments were performed to seek to improve the disorder conjugated from

conformational flexibility of the HK domain. It is known that ATP binding by the CA

domain of the histidine kinase protein induces conformational changes that lead to

trans-phosphorylation and interaction between the monomers, which may acquire a

tighter conformation (Marina et al., 2005). For that purpose, co-crystallization and

soaking experiments with ATP and non-hydrolysable ATP analogues were

performed in order to try and lock the protein into a fixed and homogeneous

conformation and to reduce the degree of disorder of the HK domain. No crystals

were obtained by the co-crystallization experiments, however, soaking experiments

with Ks-Amt5 crystals obtained in 24 % PEG 3350 + 0.1 M MES pH 7.0 lead to good

quality data collection. Regardless of that, the data collected and processed in such

crystals did not show any difference and, as it was previously described, the

histidine kinase domain was not visible. Other attempts included soaking of the Ks-

Amt5 crystals with heavy atom solutions (for MAD structure solution) (4.3.7).

However, the diffraction power of the Ks-Amt5 crystals was reduced to 8-9 Å and no

heavy atom signal for Hg, Pb, Ta, Pt, or Au was detected.

5.10 Structural comparison of Ks-Amt5 with other Amt proteins

Stuctural comparison of the integral membrane domain of Ks-Amt5 and those of the

available Amt protein structures (Ec-AmtB and Af-Amt1) revealed strong

homologies in the transmembrane region of all proteins (Figure 31A). The root

mean-squared deviations of all atoms positions without the C-terminal extension of

Ks-Amt5 are 1.08 Å between Ks-Amt5 and Ec-AmtB and 1.17 Å between Ks-Amt5

and the Af-Amt1 protein. The structural alignment shows no major differences.

However, Ks-Amt5 differs in the extension of the C-terminal with 17 amino acid

residues more than Af-Amt1 and the length of the loop 5 with 20 amino acid

residues in comparison to 13 in E. coli and 12 in Af-Amt1. In Ks-Amt5 loop 5 could be


Camila Hernández

112

involved in the interaction between monomers as mentioned. Therefore it is likely

that the length of the loop increases the possibility of contact between the loop and

the C-terminal extension.

All three structures, show two vestibules, one extracellular and one intracellular,

corresponding to the position of the recruitment site and the exit of the channel,

respectively (Figure 31C). In the extracellular vestibule of Ks-Amt5, conserved

residues W144 and S227 suggested as forming the recruitment of ammonium are

present in almost identical conformations as in Ec-AmtB and Af-Amt1 (Figure 31B).

Two additional MES molecules were identified in the extracellular vestibule at the

entrance of the hydrophobic pore, eventually blocking the substrate passage by the

formation of electrostatic interactions with the surrounding amino acids Q100,

L147, F164, L228, L229, Y345 and including W144 and S227 (Figures 32A and 32B).

In both the structures Ec-AmtB and Af-Amt1, the hydrophobic pore was visible

however, in the Ks-Amt5 it was closed. Ks-Amt5 presents two non-conserved

phenylalanine residues (F27 and F31 from helix TM1) that were found towards the

hydrophobic pore possibly blocking the channel due to steric impositions. This fact,

in addition to the side chain of an aspartate residue (D406) located at the C-

terminus, which seems to obstruct the cytoplasmic exit of the channel, could explain

the discontinuity in the hydrophobic pore (Figure 33).


Camila Hernández

113

Figure 31: Structural comparison of Ks-Amt5 with Ec-AmtB and Af-Amt1 Amt proteins. Ks-

Amt5 is represented in silver, Af-Amt1 in blue and Ec-AmtB in orange. A. The overall core of Ks-Amt5

is highly conserved to Af-Amt1 (r.m.s.d 1.17 Å) and Ec-AmtB (r.m.s.d 1.08 Å). B. Detailed side view of

the protein lumen in the aligned structures shown in A. The amino acids supposedly involved in

ammonium translocation are numbered according to the Ks-Amt5 sequence. These amino acids

represent slight variations, His-pair is shifted 1.5 Å in respect to Af-Amt1 and Phe103 presents a 40˚

tilt respect to the same residue in Af-Amt1. C. Surface representation revealing the overall protein

shape and the hydrophobic substrate passages that in Ks-Amt5 appears closed, although the

structures are highly similar, the hydrophobic substrate passage in Ks-Amt5 is closed. Additional

features of the structure are the presence of two MES molecules in the periplasmic vestibule and two

phenylalanines not conserved in Af-Amt1 and Ec-AmtB. The membrane is represented in grey.


Camila Hernández

114

Figure 32: Top view of the periplasmic vestibule of the Ks-Amt5 structure. A. Two MES

molecules (represented as sticks) were found blocking the entrance of the hydrophobic pore. The

surface of the protein is represented in light-blue. B. Detail of A without surface representation. The

entrance to the hydrophobic pore is blocked in Ks-Amt5 due to the interaction of the MES molecules

with the surrounding amino acids including the conserved W144 and S227 of the recruitment site.

Figure 33: Interaction between two Ks-Amt5 monomers. The cytoplasmic exit of the hydrophobic

channel of monomer A is blocked by the C-terminal extension of monomer B. The residue D406 is

shown as a red surface and faces the exit of the channel.


Camila Hernández

115

5.11 Small Angle X-ray Scattering

Small angle X-ray scattering (SAXS) is a technique that provides high-precision

information with respect to size and shape of molecules (Neylon, 2008). Small-angle

solution scattering does not provide information on atomic coordinates as in X-ray

crystallography, thus it is often described as a low-resolution technique. In SAXS the

rotational averaging of the molecules in solution is what limits the information

content of small-angle scattering more than the resolution limits of the experiment.

The resolution limits in a SAXS experiment are referred to in terms of the smallest

angles for which data can be measured. For the accurate interpretation of scattering

data in terms of structural parameters, it is necessary that the scattering signal is

measured from a sample composed by monodisperse and identical particles

(Jacques & Trewhella, 2010). Therefore, the sample preparation is crucial.

SAXS is generally used to study protein complexes. In addition, this technique is also

informative when one component of a protein complex is expected to undergo a

conformational change upon binding. Due to the fact that small-angle scattering

uses molecules in solution, the data are time and ensemble-averaged (Jacques &

Trewhella, 2010). Therefore, SAXS can be also used to study flexible systems.

In collaboration with Prof. Dmitri Svergun (EMBL-DESY Hamburg, Germany), small

angle X-ray scattering (SAXS) experiments were carried out with the Ks-Amt5

(pET21a::amt5) and the Ks-Kin (pET28a::kin) proteins in solution in order to obtain

a lower resolution structure with the overall shape of the protein. For both proteins,

preliminary data was obtained. However, in the case of Ks-Amt5 the scattering data

was not processable due to the interference of the detergent molecules that are

present in the buffer solution and which are necessary to stabilize the protein. On

the other hand, SAXS data for Ks-Kin was further processable and indicated two

different oligomeric states for this domain in the presence or absence of non-

hydrolysable ATP analogues (AppCp, ATPS, ATPS). Ks-Kin in absence of ATP

presented a dimeric form as it was observed with SEC and which corresponds to the

oligomerization state of other histidine kinase homologues. In the presence of the


Camila Hernández

116

non-hydrolysable ATP analogues (AppCp, ATPS, ATPS) tested, the Ks-Kin protein

showed a monomeric state that might indicate different conformations of the

protein upon binding of ATP. However, without the Amt domain it is not straight

forward to explain how these differences occur. Therefore, further optimizations

regarding sample preparation, especially for the full length protein, are needed.

5.12 Functional studies

5.12.1 Thermodynamic characterization of Ks-Kin

It is known that histidine kinases use ATP as a nucleotide. The affinity of these

proteins to ATP appears to be conserved with KD values in the range of 100 and

200 µM (Krell et al. 2010). In order to characterize the ligand binding properties of

Ks-Amt5 isothermal tritration calorimetry (ITC) experiments were carried out.

Due to the large amounts of protein required for a single ITC experiment, only the

histidine kinase domain (Ks-Kin) was tested. Different ligands were evaluated, these

included ATP, a non-hydrolysable ATP analogue (ATPS), ADP and GTP. For each

ligand and Ks-Kin variant, two independent experiments were performed and the

standard deviations were calculated for the binding constants (Ka or Kd), the

enthalpy changes (H) and the entropy changes (S). Since only one ATP binding

site was identified on the sequence, the stoichiometry of the reaction (n) is not

shown. Table 21 summarizes the results obtained.


Camila Hernández

117

Figure 33: Titration curve of Ks-Kin with various ligands and simulation profiles. For all ITC

experiments, 282 µl ATP (1.3 mM stock) were titrated to 1.4 ml Ks-Kin (0.1 mM stock) in 21

injections at 20 ˚C. The reference titration was carried out by titrating ATP to buffer without protein

under the same conditions. The data was fit in Origin using the one set of sites model after

subtracting the reference data. A. Binding of ATP. B. No binding of GTP. C. Low affinity for ADP with

no detectable binding parameters.

Table 21: Thermodynamic parameters of different ligands with Ks-Kin wild type and variants

Protein Ligand Ka [mM-1] Kd [µM] H [kcal/mol] S

[cal/mol/K] Ks-Kin ATP 123.00 7.48 8.13 0.13 -8.29 0.97 -4.98 Ks-Kin ATPS 61.00 11.00 16.39 0.09 -1.26 0.58 -21.20 Ks-Kin ADP nd nd nd nd Ks-Kin GTP nd nd nd nd

Ks-KinH460A ATP 12.64 1.08 79.11 0.93 -8.63 0.42 -16.40 Ks-KinH460A ATPS 33.60 1.09 29.76 0.92 -10.89 0.13 -10.70

Ks-KinI612W/S666W ATP nd nd nd nd

The binding of ATP to Ks-Kin is described as an exothermic reaction with an

association constant (Ka) of 123 mM-1 (Figure 33A). It was expected that the affinity

for ATP is higher than for other ligands due to the fact that ATP is the preferred


Camila Hernández

118

substrate of protein histidine kinases defined by the phenylalanine and glycine

residues of the F and G-boxes present in the binding pocket.

The binding affinity for ATPS was lower than for ATP ( Ka = 63 mM-1). This could be

explained by the weaker electronegativity of the sulfur atom in the gamma position

of the phosphate group and that non-hydrolysable properties of this ligand lead to a

weaker interaction of the ATPS to the binding pocket.

The results of ICT experiments showed non-detectable binding for GTP (Figure

33B). This result confirms that Ks-Kin as other histidine kinases like CheA or EnvZ

does not bind other nucleotides such as GTP and strongly supports the substrate

specificity of Ks-Kin like other histidine kinases for ATP. Interestingly Ks-Amt5

exhibits a very low affinity for ADP (Figure 33C) in contrast to other kinases like

CheA with a KD of 90 µM (Tawa & Steart, 1994). The ITC profile showed small

changes in the curve, with a small increase of H upon titration. It is feasible that

under these conditions, the binding was non-detectable, due to the lower

concentration of protein used (0.1 mM). However, to study this further, it is

required to increase the concentration and the protein-ligand ratio in order to

obtain suitable data for ITC simulations.

In the case of Ks-KinH460A a ten times lower binding affinity for ATP was observed.

This result suggests that the ATP binding event is strongly related to the presence of

the phosphorylation site in Ks-Am5. Furthermore, using ITC experiments the double

mutant of Ks-Kin I612W/S666W was proven to sterically interfere with the ATP

binding. The binding of ATP to Ks-Kin I612W/S666W was undetectable as well.

5.12.2 Phosphorylation analysis of kinase activity of Ks-Amt5

In addition to the ATP binding properties of Ks-Amt5, the autophosphorylation

activity was evaluated. Ks-Amt5 presents a histidine phosphorylation site identified

as H460 according to sequence analysis (5.1). In order to demonstrate the activity of

the protein, a radioactive assay was performed (4.2.3.8). For the detection of


Camila Hernández

119

radioactive signals due to protein phosphorylation, different optimization steps

were carried out. First attempts using Ks-Amt5 purified in the original SEC buffer

containing 0.03 % of DDM and incubation with radioactive [ -32P]-ATP showed no

distinguishable bands on the SDS gels. Therefore, detergent-free buffer was used for

subsequent experiments.

In order to optimize the radioactive assay, different variables were examined. For

both proteins, common parameters tested included the concentration ratio of

radioactive [ -32P]-ATP against protein, the concentration of non-radioactive ATP,

which is used to complement the overall ATP concentration and avoid overexposure

to radioactive material, and the concentrations of magnesium or manganese used as

a cofactor to stabilize the ATP molecules. Other parameters were considered such as

the concentration of ammonium in the case of Ks-Amt5 and the time of reaction for

Ks-Kin.

In the case of Ks-Kin the phosphorylation signal was not affected by the

concentration of [ -32P]-ATP or the protein concentration (Figure 34A). However,

the concentrations of non-radioactive ATP and magnesium or manganese influenced

the intensity of the signal (Figures 34B and 34C). The best phosphorylation signal

was obtained with 10 µM protein (calculated as a monomer), 50 µCi of [ -32P]-ATP

(> 3000 Ci/mmol specific activity), 1 mM of non-radioactive ATP and 50 mM of a

mixture of MgCl2 and MnCl2. This condition was then used to observe the duration of

the phosphorylation event. For this, the Ks-Kin protein was incubated at different

time periods and then further analyzed by SDS-PAGE and digital autoradiography

(4.2.3.6 and 4.2.3.8). The first phosphorylated bands were observed after 1 min of

reaction. The signal was further accumulated in time with a maximum intensity

detected upon three hours of reaction (Figure 34D). As two-component signal

transduction proteins, histidine kinases are involved in a complex signaling cascade

that includes the association to a response regulator protein (Casino et al., 2010).

Upon reception of a certain stimulus, the histidine kinase is activated leading to the

phosphorylation of the histidine residue. This event triggers the activation or


Camila Hernández

120

deactivation of other proteins, which functions as a response regulator that

interacts with the histidine kinase in order to transmit the signal further into the

transduction pathway (Casino et al., 2010). In the case of Ks-Kin the accumulation of

phosphorylation signals upon reaction with ATP (up to 3 hours) could be explained

due to the fact that in the performed in vitro assay the response regulator protein

was not incorporated. This protein would be in charge of the dephosphorylation of

the histidine kinase in order to reverse its activity.

Figure 35: Phosphorylation of Ks-Kin. The different steps for the optimization of the assay are

shown in A, B, and C. All reactions were incubated at 30 °C with buffer containing 20 mM Tris-HCl pH

8.0 plus 100 mM NaCl. A. Reactions with 1 mM MgCl2 in buffer and 200 µM ATP. B. Reactions with 50

µCi [ -32P]-ATP, 10 µl protein (calculated as monomer) and 1 mM MgCl2 in buffer. C. Reactions with

50 µCi [ -32P]-ATP, 10 µl protein (calculated as monomer) and 1 mM ATP. The best condition for the

detection of phosphorylated protein bands was further used for the determination of the reaction

time. D. Reactions with 50 µCi [ -32P]-ATP, 10 µl protein (calculated as monomer), 1 mM ATP and a

mixture of 50 mM MgCl2 + 50 mM MnCl2 in buffer. Accumulation of phosphorylated protein was

observed up to 3 hours of incubation.

The phosphorylation activity of the full-length protein (Ks-Amt5) was also analyzed.

In order to detect phospho-protein bands of Ks-Amt5, it was required to eliminate

the presence of detergents in the reaction buffer, which seemed to interfere with the

distribution of the radioactive labeled phosphate groups as only smeared bands

were obtained (data not shown). Furthermore, the size of the polymerization matrix


Camila Hernández

121

for SDS-PAGE was decreased from 12.5 % to 7.5 % polyacrylamide gels (4.2.3.6).

Upon incubation with [ -32P]-ATP, the protein aggregated. As a consequence, the

samples did not migrate in the 12.5 % resolving gel, which was generally used for

the analysis of purified Ks-Amt5.

The phosphorylation analysis of Ks-Amt5 required greater amounts of [ -32P]-ATP

and protein (figure 37A) in comparison to Ks-Kin. Further, the concentration of non-

radioactive ATP as well as magnesium and manganese played a role in the intensity

of the signal. Although high radiation intensity was detected for 50 µM ATP, 10 mM

MgCl2, and 10 mM MgCl2 + MnCl2 the protein band looked smeared and not defined

(Figures 37B and 37C). Therefore, the best condition for the detection of

phosphorylation activity for Ks-Amt5 was chosen as 30 µM protein (calculated as

monomer), 250 µCi of [ -32P]-ATP (> 3000 Ci/mmol specific activity), 1 mM of non-

radioactive ATP and 50 mM of a mixture of MgCl2 and MnCl2. This condition was

further used for the evaluation of the differences in ammonium concentration.

Ks-Amt5 was phosphorylated in the absence of ammonium and at low

concentrations of ammonium in the range of 0.5 mM to 10 mM. The phosphorylation

activity was greatly diminished at high concentrations (50 mM) and almost

completely ceased at 500 mM of ammonium (Figure 37D). These results indicate

that the concentration of ammonium influences the degree of phosphorylation of

the histidine kinase. In the absence and at lower concentrations of ammonium Ks-

Amt5 appears phosphorylated and the signal is enhanced upon the increase in the

concentration of ammonium up to 10 mM. At this point the increment in the

intensity of the signal might be explained due to the absence of a response regulator

that would acquire the phosphoryl group from the histidine kinase and as a

consequence transmits a signal further into the transduction pathway. However, the

phosphorylation signal intensity decays at higher concentrations of ammonium (50

mM and 500 mM). This fact suggests an inhibition of the Amt domain. By means of

this inhibition, the signal is no longer transmitted to the histidine kinase domain and

therefore ATP is no longer required, thus, the signaling pathway is inactivated.


Camila Hernández

122

However, since the Ks-Kin shows phosphorylation in the absence of ammonium this

is an indication that these results are artifacts of the assay and require further

evaluation.

Figure 36: Phosphorylation of Ks-Amt5. The different steps for the optimization of the assay are

shown in A, B, and C. All reactions were incubated at 30 °C with buffer containing 20 mM Tris-HCl pH

8.0, 100 mM NaCl and 10 % (v/v) glycerol. A. Reactions with 1 mM MgCl2 in buffer and 200 µM ATP.

B. Reactions with 250 µCi [ -32P]-ATP, 30 µl protein (calculated as monomer) and 1 mM MgCl2 in

buffer. C. Reactions with 250 µCi [ -32P]-ATP, 30 µl protein (calculated as monomer) and 1 mM ATP.

The best condition for the detection phosphorylated protein bands was further used for the

determination of the reaction time. D. Reactions with 250 µCi [ -32P]-ATP, 30 µl protein (calculated

as monomer), 1 mM ATP and a mixture of 50 mM MgCl2 + 50 mM MnCl2 in buffer. Upon increase in

nitrogen concentration the phosphorylation signal is enhanced, however at higher concentrations, 50

mM and 500mM, the kinase activity is diminished.

5.13 Remarks on the possible mechanism of transport for Ks-Amt5

Generally an autophosphorylation reaction requires the presence of ATP and an

external stimulus, which is detected by the sensor domain of a kinase protein. For

Ks-Amt5, the sensor domain is thought to be the integral membrane part


Camila Hernández

123

characterized as an Amt protein and whose structure was determined (5.8). As in

other Amt proteins, Ks-Amt5 has the expected amino acid residues supposed to be

involved in and required to translocate ammonium under lower nitrogen level

conditions and fulfill metabolic requirements in the cell. When extracellular

ammonium concentrations are low, ammonium could enter the cell through Ks-

Amt5 triggering a conformational change in the linker between the Amt domain and

the histidine kinase domain. Thus, the signal given by the Amt protein would induce

the activation of the histidine kinase domain, which in the presence of ATP is

phosphorylated. Upon phosphorylation of the histidine kinase domain, transport of

ammonium occurs and a signal is further transmitted to a response regulator. When

the extracellular concentrations of ammonium are high, the sensing function of the

Amt domain ceases and the histidine kinase is dephosphorylated. As a result,

transport of ammonium is inactivated as well as the signal transduction cascade. On

the other hand, the phosphorylated histidine residue could be dephosphorylated

upon interaction with a response regulator protein involved in the transmission of

the signal to the transduction pathway. The dephosphorylation event should then be

the deactivation step of the ammonium transport and thus, the key for the

regulation of the Amt protein. By means of this mechanism and due to the presented

data, Ks-Amt5 can be active only in the presence of ATP and therefore, ammonium

transport requires energy.

Since the localization of this particular protein in the cell is unknown and there are

four other Amt proteins encoded in the genome of “Ca. K. sttutgartiensis”, the real

function of the protein cannot be clarified in the present study. It is likely that the

ammonium transport mechanism is conserved since the molecular structure

obtained for the Amt domain exhibits strong homologies to its counterparts Ec-

AmtB and Af-Amt1. However, the Ks-Amt5 structure obtained was derived from

crystals in the absence of ATP. In the Ks-Amt5 structure the hydrophobic channel is

closed. This observation, in combination with the phosphorylation studies, agrees

with the fact that this particular Amt will transport ammonium only when the

histidine kinase domain is phosphorylated.


Camila Hernández

124

5.14 Future perspectives

As of yet, many structural, functional and regulatory details of the Amt/Rh protein

family have still to be revealed. Besides the transport features, evidence also shows

these proteins as ammonium sensors. It is still unclear how the signal is transferred

and which are the factors that regulate their activity. In Ec-AmtB the complex

formation with the PII protein GlnK plays a crucial role in the regulation of the

ammonium transport. Other processes that could also regulate the activity of these

proteins involve allosteric changes in the C-terminal region and phosphorylation

events. The structure of Ks-Amt5 strongly supports the similarities between the

members of the Amt family. In addition, it suggests the conservation of the

ammonium transport mechanism among the members of this protein family.

However, it is likely that in Ks-Amt5 the phosphorylation of the histidine kinase is

required for transport.

Despite the high resolution structure and the indication of the existence of a kinase

activity by Ks-Amt5, the obtained results do not explain how both domains are

organized together. The lack of a molecular structure model for the Ks-Kin domain

leads to more questions regarding possible rearrangements that might promote

transport or inactivation of the Amt sensor domain. So far, the presented results

indicate that the Ks-Kin domain works like any other kinase in the presence of ATP.

The Ks-Kin is an active protein able to bind ATP and to carry out an

autophosphorylation reaction. A change in the extracellular ammonium

concentration that can be detected by the Amt domain is thought to be the real

stimulus for the occurrence of phosphorylation. However, in the current study, the

phosphorylation of Ks-Kin occurs regardless of an external stimulus. Therefore, it

will be necessary to design new experiments to answer this problem. Experiments

with the protein reconstituted into proteoliposomes could be an alternative to

evaluate the transport activity of Ks-Amt5; nevertheless, they must be carried out


Camila Hernández

125

under conditions where the kinase activity can also be monitored. Moreover, based

on the structural knowledge of Ks-Amt5, additional constructs could be designed for

crystallization trials. In this case, mutational studies could also assess the problem

of flexibility. Double mutants for the phosphorylation site in combination with the

ATP binding site or variants completely lacking kinase activity, could be more

suitable for crystallization due to a decrease in the potential conformational changes

related to the ATP binding and phosphorylation reaction. Additional experiments

could include the optimization of samples for SAXS experiments for the full-length

protein. By means of these experiments knowledge on the overall shape of the

protein could be gained. In conclusion, the study of this interesting protein is still

open to further analysis and debate.

Appendix

Camila Hernández

126

6 Appendix

6.1 Abbreviations

2-OG 2-oxoglutarate ADP Adenosine diphosphate Amt Ammonium transport protein AppCp Adenosine-5’-[(β,γ)-methyleno]triphosphate, Sodium salt APS Ammonium persulfate ATP Adenosine triphosphate ATPS Adenosine-5’-O-(3-thio triphosphate) ATPαS Adenosine-5'-(α-thio)-triphosphate BCA Bicinchoninic acid BCIP 5-bromo-4chloro-3-indolylphosphate Bis-tris Bis(2-hydroxyethyl)-amino-tris(hydroxymethyl)-methane BN-PAGE Blue native polyacrylamide gel electrophoresis bp Base pairs CA catalytic domain of a histidine kinase CBB Coomasie brilliant blue CF 5-carboxyfluorescein CMC Critical micellar concentration ddH2O Double deionized water D9M n-nonyl-β-D-maltopyranoside D10M n-decyl-β-D-maltopyranoside D11M n-undecyl-β-D-maltopyranoside DDM n-dodecyl-β-D-maltopyranoside D13M n-tridecyl-β-D-maltopyranoside DESY Deutsches Elektronen-Synchrotron DHp Dimerization domain of a histidine kinase d TP’s deoxy-nucleoside-triphosphate DNA Deoxyribonucleic acid EDTA (Ethylenedinitrilo)tetraacetic acid EMBL European Molecular Biology Laboratory GC base pair of guanine paired with cytosine GTP Guanosine triphosphate HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HK Histidine kinase protein ITC Isothermal titration calorimetry IPTG isopropyl β-D-1-thiogalactopyranoside LDAO lauryl dimethylamine n-oxide LB Luria-Bertani

http://en.wikipedia.org/wiki/Electrophoresis

http://en.wikipedia.org/wiki/Guanine

http://en.wikipedia.org/wiki/Cytosine

Appendix

Camila Hernández

127

MA methylamine MAD multiwavelength anomalous dispersion MES 2-(N-morpholino)ethanesulfonic acid MR Molecular replacement NBT nitrotetrazolium blue OD600nm optical density at a wavelength of 600 nm OGP n-octyl-beta-D-glucopyranoside PCR Polymerase chain reaction PDB The RCSB Protein Data Bank PEG polyethylene glycol PDVF polyvinylidendifluoride r.m.s.d. root-mean-square deviation Rh Rhesus protein RNA ribonucleic acid SDS sodium dodecyl sulfate SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis

SEC Size exclusion chromatography

TAE Tris-acetate-EDTA TCS Two-component signal transduction system TEMED N,N,N`,N`-Tetramethylethylenediamine Tris tris(hydroxymethyl)aminomethane UV ultraviolet v/v Volume per volume w/v Weight per volume x g Times gravity

6.2 Units

% percentage *g multiple of gravitational acceleration ° degree °C degree Centigrade A ampere Å Angstrom (1 Å = 10-10 m) Au absorption unit Da Dalton g gram h hour L liter M molarity m meter min minute

http://en.wikipedia.org/wiki/Sodium_dodecyl_sulfate

http://en.wikipedia.org/wiki/Sodium_dodecyl_sulfate

http://en.wikipedia.org/wiki/Electrophoresis

http://dict.leo.org/ende?lp=ende&p=5tY9AA&search=gravitational

http://dict.leo.org/ende?lp=ende&p=5tY9AA&search=acceleration

Appendix

Camila Hernández

128

psi pounds per square inch (1 psi ≈ 0.07 bar) s second V volt

6.3 Prefixes

k kilo (103) c centi (10-2) m mili (10-3) μ micro (10-6) n nano (10-9) p pico (10-12)

6.4 Amino acids

A Ala alanine M Met methionine C Cys cysteine N Asn asparagine D Asp aspartate P Pro proline E Glu glutamate Q Gln glutamine F Phe phenylalanine R Arg arginine G Gly glycine S Ser serine H His histidine T Thr threonine I Ile isoleucine V Val valine K Lys lysine W Trp tryptophane L Leu leucine Y Tyr tyrosine

Appendix

Camila Hernández

129

6.5 Ks-Amt5 DNA sequence

1 ATGGAAAACATACAAATAAATATTAACCATTTGTGGGTGATTATGGCGGC 50

51 CTGCATGGTATTCTTGATGCAGTTGGGTTTTACCTCTTACGAAACCGGAT 100

101 TTTCCCAGTCCAAAAATGCCATCAGTATTGCATTGAGAAATCTCGTAGAT 150

151 ACCCTTATCTCATCACTCGTTTTTTTCAGTGTGGGCTTTGGGTTCATGTT 200

201 TGGCAAAAGCTACATGGGATTGATCGGAATAGATCTTTTCTTCGCAAATG 250

251 ATTTGGCATTGCATCCCAATACGTTATCGTATTCATTCTTTTTTTTCCAA 300

301 ATGGTCTTTGCATCCACAGCCGCCACAATATTAACAGGCGCCATAGCAGA 350

351 ACGCTCCGGTTTTATTCCCAATATAGCAGGTACCGCATTTATTGTTGCCA 400

401 TTATCTATCCAATCTTCGGGCACTGGGCATGGGGCAATCTCTTTTCCCCT 450

451 GATCAAACCGGCTGGTTAAAAGAATTGGGTTTTATTGATTTTGCAGGTGC 500

501 AACGGTAGTACATTCCATCGGCGGCTGGTTTGCCATGGCGGCGGCTATAA 550

551 TGGTAGGGCCAAGAATAGACAAATACAATCCTGACGGATCTTCTAACCGG 600

601 ATTGGGTTACATAATGTACCACTAGCCACATTAGGCACTTTTTTTCTGTG 650

651 GTTTGGTTGGTTTGGTTTTAACGGCGGAAGTCTTTTGAGAGTGAGCGTAA 700

701 ATATCGGATTGGTAATCCTGAATACGAACATGGCCGCCGCCTCTGCCGGG 750

751 GTTTCCGCCCTCATATTTATTTATGCAACAAGAAAAAGGATCGAAGCAGG 800

801 AAGTCTCTTCACTGCGATACTTGCCGGATTAGTTGCCATAACGGCAAGTT 850

851 CAAATATGGTTACCCCAGTCAGCGCAGTAGCTATCGGCCTCATTACCGGC 900

901 ATACTGGCAATCATTGCAGAAGGTTTTATTGAAAAGACTTTGAAAATCGA 950

951 CGACCCCGTAAGCGCCATTGCCGTGCACGGAGTCGGCGGGGTAATAGGTA 1000

1001 CGCTCTGCGTCGCAATATTTGCGCAAAAATCGTATCTTCTTGCGGAAAAC 1050

1051 GGAAGCAGAATGCATCAGTTAGGCATACAGGCGTTAGGCGTTATCGTCGC 1100

1101 CTTTTCATGGTCATTCGGGCTGGGCATGCTTTTCTTCTTGTGCCTAAAGA 1150

1151 AAGTAAAGAGATTACGGGTAACCCCTGAAGAAGAAAAGAGAGGACTGAAT 1200

1201 GTCGCCGAATATGAAGACGTTGCGTCGTGGCTTGATTTTATAAGAATAAC 1250

1251 ACGGCTGCAGGATATAAATACAATACTTGAAAAAAGGGTGCAGGAAAAGA 1300

1301 CAGCAGACCTTCAAATGGCAAATGTTGCTTTAGAAAAGGCAAACAGGCTG 1350

1351 AAATCTGAATTCCTGACAACAATGTCACATGAGCTGCGCACTCCTTTAAA 1400

1401 CGCAATCATTGGATTCGCAGAAGTCTTACGCGACGAAATCGCCGGTTCTC 1450

1451 TCAGCAAAGACCAAAAAGAATACGTAACCGATATTCACAGCAGCGGCCAT 1500

1501 CATCTGCTTGATATGATTAACAACATATTGGACCTTTCAAAAATTGAAAC 1550

1551 GGGGAAAATGCATCTTCAATACGAGGAATTTTGCATTGAAGATGCAATTA 1600

1601 ATGACACACTGACAATTATAAACGCATCCGCCAACAATAAAGGAATTTCC 1650

1651 GTTCATACAAATATACAGGATAACACGCCACTGCTATCCGCTGACAAAAC 1700

1701 AAAATTCAGGCAGATTCTTTATAATTTGCTATCAAATGCAGTGAAATTTA 1750

1751 CCCCTGAAAATGGCAAAATTACTATAAACGTTTTCCAAAAAGACAACTCT 1800

1801 CTGCAATTTGAAATAGTTGATACCGGCATTGGTATAAAGCCTGAAGACAA 1850

1851 AGAGAAATTATTCGAAGCATTTCACCAGGCAGATGCATCGCTTACAAGAG 1900

1901 AATATGAGGGTACAGGGCTTGGATTGCATCTGACAAAACGTCTTGTAGAA 1950

1951 TTACATGGTGGCAAGATATGGGCAGAAAGTACCTTTGGAAAAGGAAGCAC 2000

2001 CTTCTTTTTTATCTTGCCCATAAATCCAGTGAACAAG 2037

Appendix

Camila Hernández

130

6.6 Ks-Amt5 amino acid sequence

The C-terminal histidine kinase domain is highlighted in blue

1 MENIQININHLWVIMAACMVFLMQLGFTSYETGFSQSKNAISIALRNLVD 50

51 TLISSLVFFSVGFGFMFGKSYMGLIGIDLFFANDLALHPNTLSYSFFFFQ 100

101 MVFASTAATILTGAIAERSGFIPNIAGTAFIVAIIYPIFGHWAWGNLFSP 150

151 DQTGWLKELGFIDFAGATVVHSIGGWFAMAAAIMVGPRIDKYNPDGSSNR 200

201 IGLHNVPLATLGTFFLWFGWFGFNGGSLLRVSVNIGLVILNTNMAAASAG 250

251 VSALIFIYATRKRIEAGSLFTAILAGLVAITASSNMVTPVSAVAIGLITG 300

301 ILAIIAEGFIEKTLKIDDPVSAIAVHGVGGVIGTLCVAIFAQKSYLLAEN 350

351 GSRMHQLGIQALGVIVAFSWSFGLGMLFFLCLKKVKRLRVTPEEEKRGLN 400

401 VAEYEDVASWLDFIRITRLQDINTILEKRVQEKTADLQMANVALEKANRL 450

451 KSEFLTTMSHELRTPLNAIIGFAEVLRDEIAGSLSKDQKEYVTDIHSSGH 500

501 HLLDMINNILDLSKIETGKMHLQYEEFCIEDAINDTLTIINASANNKGIS 550

551 VHTNIQDNTPLLSADKTKFRQILYNLLSNAVKFTPENGKITINVFQKDNS 600

601 LQFEIVDTGIGIKPEDKEKLFEAFHQADASLTREYEGTGLGLHLTKRLVE 650

651 LHGGKIWAESTFGKGSTFFFILPINPVNK 679

References

Camila Hernández

131

7 References

Andrade, S.L.A., Dickmanns, A., Ficner, R. & Einsle, O. (2005). “Crystal structure of the archaeal ammonium transporter Amt-1 from Archaeoglobus fulgidus”. Proc. Natl. Acad. Sci. USA. 102: 14994–14999. Andrade, S.L.A., Dickmanns, A., Ficner, R. & Einsle, O. (2005b). “Expression, purification and crystallization of the ammonium transporter Amt-1 from Archaeoglobus fulgidus”. Acta. Cryst. F. 61: 861-863. Andrade, S.L.A. & Einsle, O. (2007). “The Amt/ ep/ h family of ammonium transport proteins”. Mol. Membr. Biol. 24: 357–365. Aravind, L. & Ponting, C.P. (1999). “The cytoplasmic helical linker domain of receptor histidine kinase and methyl-accepting proteins is common to many prokaryotic signalling proteins”. FEMS Microbiol. Lett. 176: 111–116. Arcondéguy, T., Jack, . & errick, . (2001). “PII signal transduction proteins, pivotal players in microbial nitrogen control”. Microbiol. Mol. Biol. Rev. 65: 80–105. Arp, . J., Chain, P.S.G. & Klotz, .G. (2007). “The Impact of Genome Analyses on Our Understanding of Ammonia-Oxidizing Bacteria”. Annu. Rev. Microbiol. 61: 503–528. Banumathi, S., Zwart, P.H., Ramagopal, U.A., Dauter, M. & Dauter, Z. (2004). “Structural effects of radiation damage and its potential for phasing”. Acta Cryst. D. 60: 1085-1093. Bao-zhen, L., Merrick, M., Su-mei, L., Hong-ying, L., Shu-wen, Z., Wei-ming, S. and Yan-hua, S. (2009). “ olecular Basis and egulation of Ammonium Transporter in ice”. Rice Sci. 16: 314-322. Beizt, E. (2000). “TeXtopo: shaded membrane protein topology plots in LaTeX2e,” Bioinformatics. 16: 1050-1051. Bendtsen, J. D., Nielsen, H., von Heijne, G. and Brunak, S. (2004). “Improved prediction of signal peptides: SignalP 3.0”. J. Mol. Biol. 340: 783-795. Berejnov, V., Husseini, N.S., Alsaied, O.A. & .E. Thorne. (2006). “Effects of cryoprotectant concentration and cooling rate on vitrification of aqueous solutions”. J. Appl. Cryst. 39: 244-251. Bertani, G. (1951). “Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli”. J. Bacteriol., 62: 293‐300.

References

Camila Hernández

132

Besant, P.G. & Attwood, P. . (2010). “Histidine phosphorilation in histones and in other mammalian proteins”. Methods Enzymol. 471: 403-426. Bilwes, A. ., Alex, L.A., Crane, B. . & Simon, .I. (1999). “Structure of CheA, a signal-transducing histidine kinase”. Cell. 96: 131–141. Bilwes, A. ., Quezada, C. ., Croal, L. ., Crane, B. . & Simon, .I. (2001). “ ucleotide binding by the histidine kinase CheA”. Nat. Struct. Biol. 8: 353–360. Blakey, D., Leech, A., Thomas, G. H., Coutts, G., Findlay, K. & Merrick, M. (2002). “Purification of the Escherichia coli ammonium transporter AmtB reveals a trimeric stoichiometry”. Biochem. J. 364: 527-535. Bostick, .L. & Brooks, C.L. (2007). “ eprotonation by dehydration: the origin of ammonium sensing in the AmtB channel”. PLoS Comput. Biol. 3: 0231-0246. Bragg, W.L. (1913) “The iffraction of Short Electromagnetic Waves by a Crystal”. Proc. Cam. Phil. Soc. 17: 43-57. Broach, J., eumann, C. & Kustu, S. (1976). “ utant strains (nit) of Salmonella typhimurium with a pleiotropic defect in nitrogen metabolism”. J. Bact. 128: 86-98. Bryson, K., McGuffin, L.J., Marsden, R.L., Ward, J.J., Sodhi, J.S. & Jones, D.T. (2005). “Protein structure prediction servers at University College London”. Nucl. Acids Res. 33: W36-W38. Brünger, A.T. (1993). “Assessment of phase accuracy by cross validation: the free value. ethods and applications”. Acta Cryst. D. 49: 24-36. Bullock, W. O., Fernandez, J. . & Short, J. . (1987). “XL1-Blue: A high efficiency plasmid transforming recA- E. coli strain with β-galactosidase selection”. Bio- Techniques 5: 376. Burnette, W. N. (1981). "Western blotting: electrophoretic transfer of proteins from sodium dodecyl sulfate polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A”. Anal. Biochem., 112: 195‐203. Cai, S.J. & Inouye, . (2003). “Spontaneous subunit exchange and biochemical evidence for trans-autophosphorylation in a dimer of Escherichia coli histidine kinase (EnvZ). J. Mol. Biol. 329: 495-503.

References

Camila Hernández

133

Casino, P., ubio, . & arina, A. (2009). “Structural Insight into Partner Specificity and Phosphoryl Transfer in Two-Component Signal Transduction”. Cell. 139: 325-336. Casino, P., Rubio, V. & arina, A. (2010). “The mechanism of signal transduction by two-component systems”. Curr. Op. Struct. Biol. 20: 763-771. Chang, C., Kwok, S.F., Bleecker, A.B. & eyerowitz, E. . (1993). “Arabidopsis ethylene-response gene ETR1: similarity of product to two-component regulators”. Science. 262: 539–544. Chiu, M.L., Tsang, C., Grihalde, N. & MacWilliams, M.P. (2008). “Over-expression, solubilization, and purification of G protein-coupled receptors for structural biology”. Comb. Chem. High Throughput Screen. 11: 439-462. CLC bio A/S. (2005). “CLC sequence viewer”. www.clcbio.com. Cochran, A.G. & Kim, P.S. (1996). “Imitation of Escherichia coli aspartate receptor signaling in engineered dimers of the cytoplasmic domain”. Science. 271: 1113–1116. Collaborative Computational Project umber 4. (1994). “The CCP4 suite: programs for protein crystallography”. Acta Cryst, Sec. D. 50: 760-763. Conroy, M.J., Durand, A., Lupo, D., Li, X.D., Bullough, P.A., Winkler, F.K. & Merrick, M. (2007). “The crystal structure of the Escherichia coli AmtB-GlnK complex reveals how GlnK regulates the ammonia channel”. Proc. Natl. Acad. Sci. USA. 104: 1213-1218. Crowther, .A. & Blow, . . (1967). “A method of positioning a known molecule in an unknown crystal structure”. Acta Cryst. 23: 544-548. Cruickshank, .W. (1999). “ emarks about protein structure precision”. Acta Cryst. D. 55: 583-601 Dauter, Z., Dauter, M. & odson, E. (2002) “Jolly SA ”. Acta Cryst. D. 58: 494-506. eLano, W.L. (2002). “The Py OL olecular Graphics System”. http://www.pymol.org. ixon, . & Kahn . (2004). “Genetic regulation of biological nitrogen fixation”. Nature Rev. Microbiol. 2: 621-631. Dodson, E. (2003). “Is it jolly SA ?”. Acta Cryst. D. 59: 1958-1965.

http://www.clcbio.com/

http://www.pymol.org/

References

Camila Hernández

134

ong, X., Stothard, P., Forsythe, I.J. & Wishart, .S. (2004). “Plas apper: a web server for drawing and auto-annotating plasmid maps”. Nucleic Acids Res. 32: W660-W664. utta, ., Qin, L. & Inouye, . (1999). “Histidine kinase: diversity of domain organization”. Mol. Microbiol. 34: 633–640. Einsle, O. & Kroneck, P.M.H. (2004). “Structural basis of denitrification”. Biol. Chem. 385: 875–883. Emsley, P. & Cowtan, K. (2004). “Coot: model building tools for molecular graphics” Acta Cryst. D. 60: 2126-2132. Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. (2010). “Features and development of Coot”. Acta Cryst. D. 66: 486-501. Evans, P. (2006). “Scaling and assessment of data quality”. Acta Cryst. D. 62: 72-82. Evans, P. & cCoy, A. (2008). “An introduction to molecular replacement”. Acta Cryst. D. 64: 1-10. Ewald, P. (1969). “Introduction to the dynamical theory of X-ray diffraction”. Acta Cryst. Sec. A. 25: 203-208. Fabret, C., Feher, V.A. & Hoch, J.A. (1999). “Two-component signal transduction in Bacillus subtilis: how one organism sees its world”. J. Bacteriol. 181: 1975–1983. Faham, S. & Bowie, J.U. (2002). “Bicelle crystallization: a new method for crystallizing membrane proterins yields a monomeric bacteriorhodopsin structure”. J. Mol. Biol. 316: 1-6. Falke, J.J. & Hazelbauer, G.L. (2001). “Transmembrane signaling in bacterial chemoreceptors”. Trends Biochem. Sci. 26: 257–265. Falkowski, P.G., Barber, .T. & Smetacek, . (1998). “Biogeochemical Controls and Feedbacks on Ocean Primary Production”. Science. 281: 200-206. Fazekas de St Groth, S., Webster, R. G. & Datyner, A. (1963). “Two new staining procedures for quantitative estimation of proteins on electrophoretic strips”. Biochim. Biophys. Acta., 71: 377-391.

References

Camila Hernández

135

Fong, . ., Kim, K.S., Yoshihara, C., Inwood, W.B. & Kustu, S. (2007). “The W148L substitution in the Escherichia coli ammonium channel AmtB increases flux and indicates that the substrate is an ion”. Proc. Natl. Acad. Sci. USA. 104: 18706–18711. Forchhammer, K. (2004). “Global carbon/nitrogen control by PII signal transduction in cyanobacteria: from signals to targets”. FEMS Microbiol. Rev. 28: 319–333. Forchhammer, K. (2008). “PII signal transducers: novel functional and structural insights”. Trends Microbiol. 16: 65-72. Fujinaga, . & ead, .J. (1987). “Experiences with a new translation function program”. J. App. Cryst. 20: 517-521. Galagan, J.E., Nusbaum, C., Roy, A., Endrizzi, M.G., MacDonald, P., FritzHugh, W., Calvo, S., Engels, R., Smirnov, S., Atmoor, D., Brown, A., Allen, N., Naylor, J., Stange-Thormann, N., DeArcellano, K., Johnson, R., Linton, L., McEwan, P., McKernan, K., Talamas, J., Tirrell, A., Ye, W., Zimmer, A., Barber, R.D., Cann, I., Graham, D.E., Graham, D.A., Guss, A.M., Hedderich, R., Ingram-Smith, C., Kuettner, H.C., Krzycki, J.A., Leigh, J.A., Li, W., Liu, J., Mukhopadhyay, B., Reeve, J.N., Smith, K., Springer, T.A., Umayam, L.A., White, O., White, R.H., Conway de Macario, E., Ferry, J.G., Jarrell, K.F., Jing, H., Macario, A.J., Paulsen, I., Pritchett, M., Sowers, K.R., Swanson, R.V., Zinder, S.H., Lander, E., etcalf, W.W. & Birren, B. (2002). “The genome of . acetivorans reveals extensive metabolic and physiological diversity”. Genome Res. 12: 532-542. Garman, E. & Owen, .L. (2006). “Cryocrystallography of macromolecules: practice and optimization”. Methods Mol. Biol. 364: 1-18. Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M. R., Appel ,R. D. & Bairoch A. (2005). “Protein Identification and Analysis Tools on the ExPASy Server”. The Proteomics Protocols Handbook. Humana Press. 571-607. Gazzarrini, S., Lejay, L., Gojon, A., Ninnemann, O., Frommer, W.B. & von Wiren, N. (1999). “Three functional transporters for constitutive, diurnally regulated, and starvation-induced uptake of ammonium into Arabidopsis roots”. Plant Cell. 11: 937-948. Green, .W., Ingram, . . & Perutz, .F. (1954). “The structure of hemoglobin. IV. Sign determination by the isomorphous replacement method”. Proc. Roy. Soc. A. 225: 287-307. Gruswitz, F., O’Connell, J. & Stroud, . . (2007). “Inhibitory complex of the transmembrane ammonia channel, AmtB, and the cytosolic regulatory protein, GlnK, at 1.96 Å”. Proc. Natl. Acad. Sci. USA. 104: 42–47.

References

Camila Hernández

136

Gruswitz, F., Chaudhary, S., Ho, J.D., Schlessinger, A., Pezeshki, B., Ho, C.M., Sali, A., Westhoff, C.M. & Stroud R.M. (2010). “Function of human h based on structure of hCG at 2.1 Å”. Proc. Natl. Acad. Sci. USA. 107: 9638–9643. Hanahan, D. (1983). “Studies on transformation of Escherichia coli with plasmids”. J. Mol. Biol., 166: 557‐580. Hendrickson, W.A., Smith, J.L., Phizackerley, R.P. & Merrit, E.A. (1988). “Crystallographic structure analysis of lamprey hemoglobin from anomalous dispersion of synchrotron radiation”. Proteins. 4: 77-88. Hess, D.C., Lu, W., Rabinowitz, J.D. & Botstein, D. (2006). “Ammonium toxicity and potassium limitation in yeast”. PloS Biol. 4: 2012-2023. Honig, B. & icholls, A. (1995). “Classical electrostatics in biology and chemistry”. Science. 268: 1144-1149. Hsing, W., usso, F. ., Bernd, K.K. & Silhavy, T.J. (1998). “ utations that alter the kinase and phosphatase activities of the two-component sensor EnvZ”. J. Bacteriol. 180: 4538–4546. Huber, . (1965). “ ie automatisierte Faltmoleku lmethode”. Acta Cryst. 19: 353-356. Javelle, A., Severi, E., Thornton, J. & errick, . (2004). “Ammonium sensing in Escherichia coli. Role of the ammonium transporter AmtB and AmtB-GlnK complex formation”. J. Biol. Chem. 279: 8530–8538. Javelle, A. & errick, . (2005). “Complex formation between AmtB and GlnK: an ancestral role in prokaryotic nitrogen control”. Biochem. Soc. Trans. 33: 170-172. Javelle, A., Lupo, D., Zheng, L., Li, X.D., Winkler, F.K. & Merrick, M. (2006). “An unusual twin-his arrangement in the pore of ammonia channels is essential for substrate conductance”. J. Biol. Chem. 281: 39492–39498. Javelle, A., Lupo, D., Li, X.D., Merrick, M., Chami, M., Ripoche, P. & Winkler, F.K. (2007). “Structural and mechanistic aspects of Amt/ h proteins”. J. Struct. Biol. 158: 472–481. Javelle, A., Lupo, D., Ripoche, P., Fulford, T., Merrick, M. & Winkler, F.K. (2008). “Substrate binding, deprotonation and selectivity at the periplasmic entrance of the E. coli ammonia channel AmtB”. Proc. Natl. Acad. Sci. USA. 105: 5040-5045. Jetten, M.S.M., Schmid, M., Schmidt, I., Wubben, M., van Dongen, U., Abma, W., Sliekers, O., Revsbech, N.P., Beaumont, H.J.E., Ottosen, L., Volcke, E., Laanbroek, H.J.,

References

Camila Hernández

137

Campos-Gomez, J.L., Cole, J., van Loosdrecht, M., Mulder, J.W., Fuerst, J., Richardson, D., van de Pas, K., Mendez-Pampin, R., Third, K., Cirpus, I., van Spanning, R., Bollmann, A., Nielsen, L.P., Op den Camp, H., Schultz, C., Gundersen, J., Vanrolleghem, P., Strous, ., Wagner, . & Kuenen, J.G. (2002). “Improved nitrogen removal by application of new nitrogen-cycle bacteria”. Rev. Environ. Sci. Biotechnol. 1: 51-63. Jetten, M.S., Cirpus, I., Kartal, B. et al. (2005a). “1994–2004: 10 years of research on the anaerobic oxidation of ammonium”. Biochem. Soc. Trans. 33: 119–133. Jetten, ., Schmid, ., van de Pas-Schoonen, K., Sinninghe- amste, J.S. & Strous, M. (2005b). “Anammox Organisms: Enrichment, Cultivation, and Environmental Analysis”. Methods. Enzymol. 397: 34-57. Jetten, M.S.M., Niftrik, L., Strous, M., Kartal, B., Keltjens, J.T., Op den Camp, H.J. (2009). “Biochemistry and molecular biology of anammox bacteria”. Crit. Rev. Biochem. Mol. Biol. 44: 1-20. Jiang, P. & Ninfa, A.J. (2007). “Escherichia coli PII signal transduction protein controlling nitrogen assimilation acts as a sensor of adenylate energy charge in Vitro”. Biochemistry. 46: 12976–12986. Jones, .T. (1999). “Protein secondary structure prediction based on position-specific scoring matrices”. J. Mol. Biol. 292: 195-202. Kartal, B., Rattray, J., van Niftrik, L., van de Vossenberg, J., Schmid, M., Webb, R.I., Schouten, S., Fuerst, J.A., Sinninghe- amste, J.S., Jetten, M.S.M. & Strous, M. (2007). “Candidatus Anammoxoglobus propionicus” gen. nov., sp. nov., a new propionate oxidizing species of anaerobic ammonium oxidizing bacteria”. Syst. Appl. Microbiol. 30: 39–49. Kartal, B., van Niftrik, L., Rattray, J., van de Vossenberg, J., Schmid, M.C., Damste, J.S.S., Jetten, M.S.M. & Strous, M. (2008). “Candidatus Brocadia fulgida”: an autofluorescent anaerobic ammonium oxidizing bacterium”. FEMS Microbiol. Ecol. 63: 46–55. Khademi, S., O’Connell, J., emis, J., obles-Colmenares, Y., Miercke, L. J. & Stroud, R. . (2004). “ echanism of ammonia transport by Amt/ EP/ h: structure of AmtB at 1.35 Å”. Science 305: 1587–1594. Khademi, S. & Stroud, . . (2006). “The Amt/ EP/ h Family: Structure of AmtB and the echanism of Ammonia Gas Conduction”. Physiology. 21: 419–429. Kleiner, . (1985a). “Bacterial ammonium transport”. FEBS Microbiol. Rev. 32: 87–100.

References

Camila Hernández

138

Kleiner, . (1985). “Energy expenditure for cyclic retention of H3/NH4+ during N2 fixation by Klebsiella pneumoniae”. FEBS Lett. 187: 237–239. Klotz, .G. & Stein, L.Y. (2008). “ itrifier genomics and evolution of the nitrogen cycle”. FEMS Microbiol. Lett. 278: 146–156. Klumpp, S., & Krieglstein, J. (2002). “Phosphorylation and dephosphorylation of histidine residues in proteins”. Eur. J. Biochem. 269: 1067–1071. Knepper, .A. (1991). “ H4+ transport in the kidney” Kidney Int. Suppl. 33: S95-S102. Krell, T., Lacal, J., Busch, A., Silva-Jimenez, H., Guazzaroni, M.E. & Ramos, J.L. (2010). “Bacterial sensor kinases: iversity in the recognition of environmental signals”. Annu.Rev.Microbiol. 64: 539-559. Krogh, A., Larsson, B., von Heijne, G. & Sonnhammer, E.L.L. (2001). “Predicting transmembrane protein topology with a hidden Markov model: Application to complete genomes”. J. ol. Biol. 305:567-580. Kuenen, J.G. (2008). “Anammox bacteria: from discovery to application”. Nature Rev. Microbiol. 6: 320-326. Kuenen, J.G. & Jetten, M.S.M. (2001). “Extraordinary anaerobic ammonium oxidizing bacteria”. A.S.M. News. 67: 456-463. Kuypers, M.M.M., Sliekers, A.O, Lavik, G., Schmid, M., Jørgensen, B.B., Kuenen, J.G., Sinninghe- amste, J.S., Strous, . & Jetten, .S. . (2003). “Anaerobic ammonium oxidation by anammox bacteria in the Black Sea”. Nature. 422: 608-611. Lacapère, J.J., Pebay-Peyroula, E., Neumann, J.M. & Etchebest, C. (2007). “ etermining membrane protein structures: still a challenge!”. TRENDS Biochem. Sci. 32: 259-270. Laemmli, U. K. (1970). “Cleavage of structural proteins during the assembly of the head of bacteriophage T4”. Nature, 227: 680-685. Lamoureux, G., Javelle, A., Baday, S., Wang, S. & Berneche, S. (2010). “Transport mechanisms in the ammonium transporter family”. Transfus. Clin. Biol. 17: 168-175. Lande, M.B., Donovan, J.M. & Zeidel, M.L. (1995). “The relationship between membrane fluidity and permeabilities to water, solutes, ammonia, and protons”. J. Gen. Physiol. 106: 67–84.

References

Camila Hernández

139

Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A., McWilliam, H., Valentin, F., Wallace, I.M., Wilm, A., Lopez, R., Thompson, J.D., Gibson, T.J. & Higgins, .G. (2007). “Clustal W and Clustal X version 2.0”. Bioinformatics. 23: 2947-2948. Laskowski, R.A., MacArthur, M.W., Moss, D.S. & Thornton, J.M. (1993). “Procheck: a program to check the stereochemical quality of protein structures”. J.Appl.Cryst. 26: 283-291. Leslie, A.G.W. (1992). “ ecent changes to the OSFL package for processing film and image plate data”. Joint CCP4 + ESF-EAMCB Newsletter Prot. Cryst. 26. Levenberg, K. (1994). “A ethod for the Solution of Certain Problems in Least Squares”. Quart. Appl. Math. 2: 164-168. Li, X., Jayachandran, S., Nguyen, H.H. & Chan, M.K. (2007). “Structure of the Nitrosomonas europaea h protein”. Proc. Natl. Acad. Sci. USA. 104: 19279–19284. Loque , D., Lalonde, S., Looger, L.L., von Wirén, N., & Frommer, W.B. (2007). “A cytosolic trans-activation domain essential for ammonium uptake”. Nature. 446: 195-198. Loque , ., ora, S.I., Andrade, S.L.A., Pantoja, O. Frommer W.B. (2009). “Pore Mutations in Ammonium Transporter AMT1 with Increased Electrogenic Ammonium Transport Activity”. J. Biol. Chem. 284: 24988–24995. Ludewig, U., von Wirén, N., Rentsch, D. & Frommer W.B. (2001). “ hesus factors and ammonium: a function in efflux?”. Genome Biol. 2: 1010.1-1010.5. Ludewig, U., von Wire n, N. & Frommer, W.B. (2002). “Uniport of H4+ by the root hair plasma membrane ammonium transporter LeA T1;1”. J. Biol. Chem. 277: 13548–13555. Ludewig, U., Wilken, S., Wu, B., Jost, W., Obrdlik, P., El Bakkoury, M., Marini, A.M., Andre, B., Hamacher, T., Boles, E., von Wiren, . & Frommer, W.B. (2003). “Homo- and hetero-oligomerization of AMT1 NH4+-uniporters”. J. Biol. Chem. 278: 45603-45610. Ludewig, U. (2006). “Ion transport versus gas conduction: function of A T/ h- type proteins”. Transfus. Clin. Biol. 13: 111–116. Lukat- odgers, G.S., & odgers, K. . (1997). “Characterization of Ferrous FixL− itric Oxide Adducts by Resonance aman Spectroscopy”. Biochemistry. 36: 4178-4187.

References

Camila Hernández

140

Lupo, D., Li, X.D., Durand, A., Tomizaki, T., Cherif-Zahar, B., Matassi, G., Merrick, M. & Winkler, F.K. (2007). “The 1.3-Å resolution structure of Nitrosomonas europaea Rh50 and mechanistic implications for NH3 trasnport by hesus family proteins”. Proc. Natl. Acad. Sci. USA. 104: 19303–19308. Maeda, T., Wurgler- urphy, S. . & Saito, H. (1994). “A two- component system that regulates an osmosensing AP kinase cascade in yeast”. Nature. 369: 242–245. Marina, A., Mott, C., Auyzenberg, A., Hendrickson, W.A. & Waldburger, C.D. (2001). “Structural and utational Analysis of the PhoQ Histidine Kinase Catalytic omain”. J. Biol. Chem. 276: 41182-41190. Marina, A., Waldburger, C.D. & Hendrickson, W.A (2005). “Structure of the entire cytoplasmic portion of a sensor histidine-kinase protein”. EMBO J. 24: 4247-4259. arini, A. ., issers, S., Urrestarazu, A. & André, B. (1994). “Cloning and expression of the MEP1 gene encoding an ammonium transporter in Saccharomyces cerevisiae”. EMBO J. 13: 3456-3463. Marini, A.M., Soussi-Boudekou, S., issers, S., & André, B. (1997). “A family of ammonium transporters in Saccharomyces cerevisiae”. Mol. Cell. Biol. 17: 4282-4293. Marini, A.M., Boeckstaens, M., Benjelloun, F., Cheri f-Zahar, B. & André, B. (2006). “Structural involvement in substrate recognition of an essential aspartate residue conserved in Mep/Amt and Rh-type ammonium transporters”. Curr. Gent. 49: 364-374. arquardt, . (1963). “An Algorithm for Least-Squares Estimation of Nonlinear Pa- rameters”. SIAM J. Appl. Math. 11: 431-441. atthews, B.W. (1968). “Solvent content of protein crystals”. J. Mol. Biol. 33: 491-497. atthews, B.W. (1976). “X-ray Crystallographic Studies of Proteins”. Ann. Rev. Phys. Chem. 27: 493-493. ayer, . & Ludewig, U. (2006). “ ole of A T1;1 in H4+ acquisition in Arabidopsis thaliana. Plant Biol. 8: 522-528. McPherson, A. (1982). “Preparation and analysis of protein crystals”. ew York: Wiley & Sons.

References

Camila Hernández

141

Meyer, T. S. & Lamberts, B. L. (1965). “Use of coomassie brilliant blue 250 for the electrophoresis of microgram quantities of parotid saliva proteins on acrylamide gel strips. Biochim. Biophys. Acta., 107: 144‐145. iroux, B. & Walker, J. E. (1996). “Over-production of proteins in E. coli: Mutant Hosts that allow Synthesis of some Membrane Proteins and Globular Proteins at High Levels”. J. Mol. Biol. 260: 289-298. Miyatake, H., Mukai, M., Adachi, S., Nakamura, H., Tamura, K., Iizuka, T., Shiro, Y., Strange, R.W. & Hasnain, S.S. (1999). “Iron coordination structures of oxygen sensor FixL characterized by Fe K-edge extended x-ray absorption fine structure and resonance aman spectroscopy”. J. Biol. Chem. 274: 23176–23184. Mouro-Chanteloup, I., Cochet, S., Chami, M., Genetet, S., Zidi-Yahiaoui, N., Engel, A., Colin, Y., Bertrand, O. & ipoche, P. (2010). “Functional reconstitution into liposomes of purified human hCG ammonia channel”. PLoS One. 5: 1-7. urshudov, G. ., agin, A.A. & obson, E.J. (1997). “ efinement of macromolecular structures by the maximum-likelihood method”. Acta Cryst. D. 53: 240-255. Nakhoul, N.L., Dejong, H., Abdulnour-Nakhoul, S.M., Boulpaep, E.L., Hering-Smith, K. & Hamm, L.L. (2005). “Characteristics of renal hbg as an H4+ transporter”. Am. J. Physiol. Ren. Physiol. 288: F170-F181. ewby, Z.E. ., O’connell III, J. ., Gruswitz, F., Hays, F.A., Harries, W.E.C., Harwood, I. ., Ho, J. ., Lee, J.K., Savage, .F., iercke, L.J.W. & Stroud, . . (2009). “A general protocol for the crystallization of membrane proteins for X-ray structural investigation”. Nature protocols. 4: 619-637. eylon, C. (2008). “Small angle neutron and X-ray scattering in structural biology: recent examples from the literature”. Eur. Biophys. J. Biophys. Lett. 37: 531-541. infa, A.J. & Atkinson . . (2000). “PII signal transduction proteins”. Trends Microbiol. 8: 172-179. infa, A.J. & Jiang, P. (2005). “PII signal transduction proteins: sensors of alpha-ketoglutarate that regulate nitrogen metabolism”. Curr. Opin. Microbiol. 8: 168–173. Ninnemann, O., Jauniaux, J.C. & Frommer, W.B. (1994). “Identification of a high affinity NH4+ transporter from plants”. EMBO J. 13: 3464-3471. u hse, T.S., Stensballe, A., Jensen, O.N. & Peck, S.C. (2004). “Phosphoproteomics of the arabisopsis plasma membrane and a new phosphorylation site database”. Plant Cell. 16: 2394-2405.

References

Camila Hernández

142

ygaard, T.P., ovira, C., Peters, G.H. & Jensen, .Ø. (2006). “Ammonium recruitment and ammonia transport by E. coli ammonia channel AmtB”. Biophys. J. 91: 4401–4412. Ota, I. . & arshavsky, A. (1993). “A yeast protein similar to bacterial two-component regulators”. Science. 262: 566–569. Parkinson, J.S. & Kofoid, E.C. (1992). “Communication modules in bacterial signaling proteins”. Annu. Rev. Genet. 26: 71–112. Patterson, A.L. (1934). “A Fourier Series ethod for the etermination of the Com- ponents of Interatomic istances in Crystals”. Phys. Rev. 46: 372-376. Perutz, . (1956). “Isomorphous replacement and phase determination in non-centrosymmetric space groups”. Acta Cryst. 9: 867-873. Pierce, . ., aman, C.S. & all, B.T. (1999). “Isothermal Titration Calorimetry of Protein-Protein Interactions”. Methods. 19: 213-221. Purich, .L. (1998). “Advances in the enzymology of glutamine synthesis”. In: Purich, D.L. (ed) Amino Acid Metabolism, pp 9-42. John Wiley &Sons, New York. Quan, Z.X., Rhee, S.K., Zuo, J.E., Yang, Y., Bae, J.W., Park, J.R., Lee, S.T. & Park, Y.H. (2008). “ iversity of ammonium-oxidizing bacteria in a granular sludge anaerobic ammonium-oxidizing (anammox) reactor”. Environ. Microbiol. 10: 3130–3139. Ramachandran, G.N. & Sasisekharan, V. (1968). “Conformation of polypeptides and Proteins”. Adv. Prot. Chem. 23: 283-438. ees, .C. & Howard, J.B. (2000). “ itrogenase: standing at the crossroads”. Curr. Opin. Chem. Biol. 4: 559-566. Rees, D. C., Tezcan, F. A., Haynes, C. A., Walton, M. Y., Andrade, S., Einsle, O. & Howard, J. B. (2005). “Structural basis of biological nitrogen fixation”. Philos. Trans. R. Soc. London. 363: 971–984. Reinelt., S., Hofmann, E., Gerharz, T., Bott, M. & Madden, D.R. (2003). “The structure of the periplasmic ligand-binding domain of the sensor histidine kinase CitA reveals the first extracellular PAS domain”. J. Biol. Chem. 278: 39189-39196. eitzer, L. (2003). “ itrogen assimilation and global regulation in Escherichia coli”. Annu. Rev. Microbiol. 57: 155–176.

References

Camila Hernández

143

Renart, J., Reiser, J. & Stark, G. R. (1979). “Transfer of proteins from gels to diazobenzyloxymethyl paper and detection with antisera: a method for studying antibody specificity and antigen structure”. Proc. Natl. Acad. Sci. USA, 76: 3116-3120. eynolds, J.A. & Tanford, C. (1970). “The gross conformation of protein-sodium dodecyl sulfate complexes”. J. Biol. Chem. 245: 5161-5165. Ripoche, P., Bertrand, O., Gane, P., Birkenmeier, C., Colin, Y. & Cartron, J.P. (2004). “Human hesus-associated glycoprotein mediates facilitated transport of NH3 into red blood cells”. Proc. Natl. Acad. Sci. USA. 101: 17222–17227. occhia, W., Alexov, E. & Honig, B. (2001). “Extending the applicability of the nonlinear Poisson-Boltzmann equation: multiple dielectric constants and multivalent ions”. J. Phys. Chem. B. 105: 6754. hodes, G. (2006). “Crystallography made crystal clear: a guide for users of macromolecular models”. Amsterdam, Oxford Academic. Rossmann, M. G. & Blow, D. M. (1962). “The detection of subunits within the crystallographic asymmetric unit”. Acta Cryst. 15: 24-31. Sakai, H., Wang, H.F., Takemoto-Hori, C., Kaminishi, T., Yamaguchi, H., Kamewari, Y., Terada, T., Kuramitsu, S., Shirouzu, . & Yokoyama, S. (2005). “Crystal structures of the signal transducing protein GlnK from Thermus thermophilus Hb8”. J. Struct. Biol. 149: 99– 110. Schägger, H. & Jagow, G. (1991). "Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form". Anal. Biochem., 199 (2): 223-231. Schmid, M., Twachtmann, U., Klein, M., Strous, M., Juetschko, S., Jetten, M., Metzger, J.W., Schleifer, K.H. & Wagner, M. (2000). “ olecular Evidence for Genus Level Diversity of Bacteria Capable to Catalyze Anaerobic Ammonium-Oxidation”. System. Appl. Microbiol. 23: 93-106 Schmid, ., Walsh, K., Webb, ., ijpstra, W.I.C., van de Pas‐Schoonen, K.T., Verbruggen, M.J., Hill, T., Moffett, B., Fuerst, J., Schouten, S., Damste, J.S.S., Harris, J., Shaw, P., Jetten, .S. ., and Strous, . (2003). “Candidatus Scalindua brodae’’, sp. nov., Candidatus Scalindua wagneri’’, sp. nov., two new species of anaerobic ammonium oxidizing bacteria”. Syst. Appl. Microbiol. 26: 529–538. Schmid, M.C., Risgaard-Petersen, N., van de Vossenberg, J., Kuypers, M.M.M., Lavik, G., Petersen, J., Hulth, S., Thamdrup, B., Canfield, D., Dalsgaard, T., Rysgaard, S., Seir,

References

Camila Hernández

144

M.K., Strous, M., Op den Camp, H.J. . & Jetten, .S. . (2007). “Anaerobic ammonium-oxidizing bacteria in marine environments: widespread occurrence but low diversity”. Environ. Microbiol. 9: 1476-1484. Schmidt, I., Bock, E. & Jetten, M.S.M. (2001). “Ammonia oxidation by itrosomonas eutropha with O2 as oxidant is not inhibited by acetylene”. Microbiology 147: 2247–2253. Schrödinger, LLC. (2009). “The Py OL olecular Graphics System, ersion 1.2r3pre”. http://www.pymol.org. Sevvana, M., Vijayan, V., Zweckstetter, M., Reinelt, S. & Madden, D.R. (2008). “A ligand-induced switch in the periplasmic domain of the sensor histidine kinase CitA”. J. Mol. Biol. 377: 512-523. Sinninghe- amste , J.S., Rijpstra, W.I.C., Strous, M., Jetten, M.S.M., David, O.R.P., Geenevasen, J.A.J. & van aarseveen, J.H. (2004). “A mixed ladderane/n-alkyl glycerol diether membrane lipid in an anaerobic ammonium-oxidizing bacterium”. Chem. Commun. 22: 2590–2591. Smith, P. K., Krohn, R. I., Hermanson, R. I., Mallia, A. K., Gartner, F. H., Provenzano, M. ., Fujimoto, E. K., Goeke, . ., Olson, B. J. & Klenk, . C. (1985). “ easurement of protein using bicinchoninic acid”. Anal. Biochem., 150: 75-85. Sonnhammer, E.L.L, von Heijne, G. & Krogh, A. (1998). “A hidden Markov model for predicting transmembrane helices in protein sequences”. In J. Glasgow, T. Littlejohn, F. Major, R. Lathrop, D. Sankoff, and C. Sensen, editors, Proceedings of the Sixth International Conference on Intelligent Systems for Molecular Biology, pages 175-182, Menlo Park, CA, 1998. AAAI Press. Soupene, E., He, L., Yan, . & Kustu, S. (1998). “Ammonia acquisition in enteric bacteria: physiological role of the ammonium/methylammonium transport B (AmtB) protein”. Proc. Natl. Acad. Sci. USA. 95: 7030-7034. Soupene, E., Lee, H. & Kustu, S. (2002). “Ammonium/methylammonium transport (Amt) proteins facilitate diffusion of NH3 bidirectionally”. Proc. Natl. Acad. Sci. USA. 99: 3926-3931. Stock, J.B., infa, A., & Stock, A. . (1989). “Protein phosphorylation and regulation of adaptive responses in bacteria”. Microbiol. Rev. 53: 450-490. Stock, A. ., obinson, .L., & Goudreau, P. . (2000). “Two-component signal transduction”. Annu. Rev. Biochem. 69: 183–215.

http://www.pymol.org/

References

Camila Hernández

145

Strous, M., Heijnen, J.J., Kuenen, J.G. & Jetten, M.S.M. (1998). The sequencing batch reactor as a powerful tool for the study of slowly growing anaerobic ammonium-oxidizing microorganisms”. Appl. Microbiol. Biotechnol. 50: 589-596. Strous, M., Fuerst, J.A., Kramer, E.H.M., Logemann, S., Muyzer, G., van de Pas-Schoonen, K.T., Webb, ., Kuenen, J.G. and Jetten, .S. . (1999). “ issing lithotroph identified as new planctomycete”. Nature. 400: 446–449. Strous, M., Pelletier, E., Mangenot, S., Rattei, T., Lehner, A., Taylor, M.W., Horn, M., Daims, H., Bartol-Marvel, D., Wincker, P., Barbe, V., Fonknechten, N., Vallenet, D., Segurens, B., Schenowitz-Truong, C., Médigue, C., Collingro, A., Snel, B., Dutilh, B.E., et al. (2006). “ eciphering the evolution and metabolism of an anammox bacterium from a community genome”. Nature. 440: 790-794. Studier, F. W. & Moffatt, B. A. (1986). “Use of bacteriophage T7 RNA polymerase to direct selective high level expression of cloned genes”. J. Mol. Biol., 189: 113-130. Stura, E. A. (1999). “ everse screening. In: Crystallization of proteins: Techniques, strategies and tips”. A laboratory manual (Bergfors T.). 113–124. Internaltional university line Stura, E. A., emerow, G. . & Wilson, I. A. (1992). “Strategies in the crystallization of glycoproteins and protein complexes”. J. Cryst. Growth. 122: 273–285. Stura, E. A., Sattertwaith, A. C., Calvo, J. C., Kaslow, D. C. & Wilson, I. A. (1994). “ everse screening”. Acta Cryst. D50: 448–455. Szczerba, M.W., Britto, D.T., Ali, S.A., Balkos, K. . & Kronzucker, H.J. (2008). “ H4+ stimulated and inhibited components of K+ transport in rice (Oryza sativa)”. J. Exp. Bot. 59: 3415-3423. Tanaka, T., Kawata, . & ukai, K. (1991). “Altered phosphorylation of Bacillus subtilis DegU caused by single amino acid changes in egS”. J. Bacteriol. 173: 5507–5515. Tanaka, T., Saha, S.K., Tomomori, C., Ishima, R., Liu, D., Tong, K.I., Park, H., Dutta, R., Swindells, M.B., Yamazaki, T., Ono, A.M., Kainosho, M., Inouye, M. & Ikura, M. (1998).“NMR structure of the histidine kinase domain of the E. coli osmosensor EnvZ”. Nature. 396: 88–92. Tawa, P. & Steart, .C. (1994). “Kinetics of CheA autophosphorylation and dephosphorylation reactions”. Biochem. 33: 7917-7924.

References

Camila Hernández

146

Taylor, B.L. & Zhulin, I.B. (1999). “PAS domains: internal sensors of oxygen, redox potential, and light.”. Microbiol. Mol. Biol. Rev. 63: 479-506. Taylor, G. (2003). “The phase problem”. Acta Cryst. D. 59: 1881-1890. Thomas, G.H., ullings, J.G.L. & errick, . (2000a). “ embrane topology of the ep/Amt family of ammonium transporters”. Mol. Microbiol. 37: 331-344. Thomas, G., Coutts, G. & errick, . (2000b). “The glnKamtB operon. A conserved gene pair in prokaryotes”. Trends Genet. 16: 11–14. Thompson, J.D., Gibson, T.J. & Higgins, .G. (2002). “ ultiple sequence alignment using ClustalW and ClustlX”. Curr. Prot. Bioinfo. Chapter 2: Unit 2.3. Tomomori, C., Tanaka, T., Dutta, R., Park, H., Saha, S.K., Zhu, Y., Ishima, R., Liu, D., Tong, K.I., Kurokawa, H., Qian, H., Inouye, M. & Ikura, M. (1999). “Solution structure of the homodimeric core domain of Escherichia coli histidine kinase EnvZ”. Nat. Struct. Biol. 6: 729–734. Towbin, H., Staehelin, T. & Gordon, J. (1979). “Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications”. Proc. Natl. Acad. Sci. USA, 76: 4350‐4354. Tremblay, P.L. & Hallenbeck, P.C. (2008). “Of blood, brains and bacteria, the Amt/ h transporter family: emerging role of Amt as a unique microbial sensor”. Mol. Microbiol. 71: 12–22. agin, A. & Teplyakov, A. (1997). “ OL EP: an automated program for molecular replacement”. J. Appl. Cryst. 30: 1022-1025. VanAken, T., Foxall-VanAken, S., Castleman, S. and Ferguson-Miller, S. (1986). “Alkyl glycoside detergents: synthesis and applications to the study of membrane proteins”. Methods Enzymol. 125: 27-35 van de ossenberg, J., attray, J.E., Geerts, W., Kartal, B., van iftrik, L., van onselaar, E.G., Sinninghe- amste , J.S., Strous, M. & Jetten, M.S.M. (2008). “Enrichment and characterization of marine anammox bacteria associated with global nitrogen gas production”. Environ. Microbiol. 10: 3120–3129. van Dommelen, A., de Mot, R. & Vanderleyden, J. (2001). “Ammonium transport: unifying concepts and unique aspects”. Aust. J. Plant Physiol. 28: 959-967.

References

Camila Hernández

147

van Niftrik, L.A., Fuerst, J.A., Damste, J.S.S., Kuenen, J.G., Jetten, M.S.M. & Strous, M. (2004). “The anammoxosome: an intracytoplasmic compartment in anammox bacteria”. FEMS Microbiol. Lett. 233: 7-13. van Niftrik, L.A., Geerts, W.J.C., van Donselaar, E.G., Humbel, B.M., Webb, R.I., Fuerst, J.A., erkleij, A.J., Jetten, .S. . & Strous, . (2008). “Linking Ultrastructure and Function in Four Genera of Anaerobic Ammonium-Oxidizing Bacteria: Cell Plan, Glycogen Storage, and Localization of Cytochrome c Proteins”. J. Bact. 190: 708-717. o gtle, F.N., Schmidt, O., Chacinska, A., Pfanner, N. & Meisinger, C. (2010). “ ative techniques for analysis of mitochondrial protein import. Methods Mol. Biol. 619: 425-436. von Heijine, G. & Gavel, Y. (1988). “Topogenic signals in integral membrane proteins” Eur. J. Biochem. 174: 671-678. von Wirén, N., Gazzarrini, S., Gojont, A. & Frommer, W.B. (2000). “The molecular physiology of ammonium uptake and retrieval”. Curr. Opin. Plant Biol., 3: 254-261. Weiss, M.S., Sicker, T., Djinovic-Carugo, K. & Hilgenfeld, R. (2001). “On the routine use of soft X‐rays in macromolecular crystallography”. Acta Cryst. D. 57: 689-695. West, A.H. & Stock, A.M. (2001). “Histidine kinases and response regulator proteins in two-component signaling systems”. Trends Biochem. Sci. 26: 369-376. Williams, S.B. & Stewart, . (1999). “Functional similarities among two-component sensors and methyl-accepting chemotaxis proteins suggest a role for linker region amphipathic helices in transmembrane signal transduction”. Mol. Microbiol. 33: 1093–1102. Wiseman, T., Williston, S., Brandts, J. & in, L. (1989). “ apid measurement of binding constants and heats of binding using a new titration calorimeter”. Anal. Biochem. 179: 131-137. Wittig, I., Braun, H.P. & Schägger, H. (2006). "Blue native PAGE". Nat. Protoc. 1 (1): 418-428. Xiao, B. & Gamblin, S.J. (1996). “The effects of cryoprotectant on crystal stability”. J. Cryst. Growth. 168: 244-247. Xu, Y.B., Cheah, E., Carr, P.D., van Heeswijk, W.C., Westerhoff, H.V., Vasudevan, S.G. & Ollis, .L. (1998). “GlnK, a PII-homologue: structure reveals ATP binding site and indicates how the T-loop may be involved in molecular recognition”. J. Mol. Biol. 282: 149–165.

References

Camila Hernández

148

Ye, .W. & Thomas, S. . (2001). “ icrobial nitrogen cycles: physiology, genomics and applications”. Curr. Op. Microbiol. 4: 307-312. Yildiz, O., Kalthoff, C., aunser, S. & Ku hlbrandt, W. (2007). “Structure of GlnK1 with bound effectors indicates regulatory mechanism for ammonia uptake”. EMBO J. 26: 589–599. Zheng, L., Kostrewa, D., Berneche, S., Winkler, F. K. & Li, X. D. (2004). “The mechanism of ammonia transport based on the crystal structure of AmtB of Escherichia coli”. Proc. Natl. Acad. Sci. USA. 101: 17090–17095. Zumft, W. G. (1997). “Cell biology and molecular basis of denitrification”. Microbiol. Mol. Biol. Rev. 61: 533–616.

Acknowledgements – Danksagung - Agradecimientos

Camila Hernández

149

8 Acknowledgements – Danksagung – Agradecimientos

This work was carried out in the Department of Molecular and Structural Biology of the

University of Göttingen and the Department of Biochemistry of the University of Freiburg

with the financial support of the German Academic Exchange Service (DAAD – Deutscher

Akademischer Austausch Dienst) to Camila Hernández.

When people read the thesis acknowledgements of someone they know, they just want to see

their names written there. I must admit that everybody has an influence during the time of

your work one way or the other, although some others have an influence just always. In my

case the list of people that I would like to thank is enormous and for that I am really grateful. I

will try to mention them all but I apologize in advance if I forgot your name…

First of all I want to thank my supervisor Dr. Susana Andrade for giving me the opportunity

to start with this challenging topic without any previous experience. Thank you for the all

the interesting discussions and the support throughout the whole process. I want to thank

Prof. Oliver Einsle for taking the position as co-supervisor and for all the good ideas and

discussions regarding this work. In addition I want to thank you both for all the good times

outside and inside the lab, thank you for your advice and the knowledge that you gave me.

I would also like to thank Prof. Andreas Bechthold for accepting the position as my third

examiner.

My deepest and sincere gratitude goes to LiWi for caring support all over my time in

Freiburg. You are like my second mother. Thank you for taking care of me and for giving me

many nice advices at any time. You are an extraordinary person and I am deeply in your

gratitude.

Martita, gracias and thank you for all the good times, for listening whenever needed and for

being yourself. It was a very nice experience to share very funny times with you and I hope

that we still have many, many more.

I would like to thank Sanjana for all the help in the radioactive lab. Thank you for the tricks

as well for the quick chats and talks in the lab which made it nice working atmosphere.

I want to thank my former colleagues Daniel C and Volodimir for all the good times in the

lab and help in the lab. Especially to Volodimir I want to thank for introducing me into the

field of radioactivity. To Heng Keat and Sohail I want to thank for the nice conversations and

times expend in the lab.

Fur alles dass ich in diesem vier Jahre gelernt habe, will ich Euch auf Deutsch danken.

Besonderen Dank geht an Frau Metje, für immer dabei sein und für die Hilfe wegen mein

DAAD Stipendium.


Camila Hernández

150

Von meiner Zeit in Göttingen danke ich alle Leute von der Arbeitskreis von Prof. Ralf Ficner,

Kristina, Sarah H., Anette, Angela und besonderen dank gilt hierbei Chrissoula für die

schöne gemeinsame Zeit.

Mein besonderer Dank gilt an alle meine Mitarbeitenpranktikanten, Emmanuel, Anika, Maid,

Melanie und Oliver. Danke für die nette Zeit im Labor, ich habe von euch auch gelernt.

Ein herzlichen Danke geht an meine lieben Tobis, (Tobi P und Tobi W), nicht nur für die

schöne Zeit im Labor, sondern auch für eure immer gute Laune und die vielen interessanten

Diskussionen und Hilfestellungen und netten Ratschläge.

Herzlicher Dank geht an alle leute in der AG Andrade, AG Einsle und AG Friedrich, Paula,

Phillip, Andrea, Sergej. Eva, Lisa, Anja W, Florian, Daniel S., Nikola, Stefan S, Marius, Heiko,

Katarina, Klaudia, für die freundliche und gute Arbeitsatmosphäre. Ein besonderes

Dankeschön gilt Wohli, für die Tips und Tricks bei verschiedenen Äkta-Problemen und für

die nette Zeit in and außerhalb des Labors. Vor allem Frau Weiser, Elke, Toni, Christiane,

Angelika möchte ich ganz speziell danken, für eure Hilfe zu jeder Zeit.

Ich danke auch an allen ehemalige Mitarbeiter dass ich kennen gelernt habe, David, Ed, Jan,

Felix, Sarah M, Daniel B, Antonia, ins besonderes an Daniel H für die Tips, Tricks und

allegemeine Zeit, Herr Hamacher, Claudia für die schöne Zeit im Labor und allgemein für die

netten freundlichen Diskussionen.

Stefan, CHICO!!! Du allein solltest 100 Seiten hier bekommen. Ohne dich wäre diese Arbeit

nicht möglich. Danke für die Tips und Tricks im Labor und für deine stetige Hilfe. Ich danke

dir sehr für alle Momente im Labor und außerhalb der Arbeit und für dein Verständnis

allermöglichen Dinge. GRACIAS.

Ein GANZ HERZLICHER und besonders LIEBER Dank geht an meine Freunde Anja, Ramona,

Peer, Wei, Juan, Julian, Thomas, Bianca, Sandra H - ohne euch wäre diese Arbeit niemals

fertig. Danke für die Ratschläge, für die Tips, Tricks und vor ALLEM die vielen besonderen

und unvergesslichen Momenten. Ich liebe euch sehr.

Ein herzlicher und extrem LIEBER Dank geht an D für die Korrektur dieser Arbeit, für alle

Diskussionen und Ratschläge. Auch für die Tolle Musik und die nette und schöne Zeit

während der Schreibphase und mancher Insomnia. Du weisst schon was es bedeutet -

Fünftausendmal r a w r.

Y como dicen por allí, los últimos siempre serán los primeros…

Gracias a todos los miembros de la familia gárgola (Hortensia, Gerardo, Fabián, Sandra,

Vivian, Elkin, Olga, Otto, Marisa, Alex, Paco, MariJuli y Johnny) por todos los buenos

momentos compartidos y en especial por el apoyo en las buenas y en las malas, por los

consejos, por escuchar y por estar presentes en los momentos de añoranza. Los quiero

mucho.


Camila Hernández

151

Carlos, gracias por todo tu apoyo y por todos estos años que compartimos juntos. Sin tí no

hubiera llegado hasta donde estoy hoy. Nuevamente mil gracias, fue una grandiosa e

inolvidable experiencia.

A mi familia y en especial a todos mis tíos y tías quienes siempre estuvieron de alguna

manera pendientes. En particular, gracias a José Frank y Loida por todo el cariño y apoyo.

Los quiero un montón.

A todos mis amigos que a pesar de la distancia siempre estuvieron allí pendientes de mí,

Isis, Hector, Ariany, Lorena, Marialex, Carolina, Yornayser, Hermes, Gaby, Mauro. Los quiero

y extraño un monton.

Madre, no tengo palabras suficientes para agradecerte todo lo que haz hecho por mí. Gracias

por estar allí en todo momento, por ser mi amiga y consejera, por tu paciencia y sobre todo

por las buenas vibraciones que llegaron a mí cuando más lo necesitaba. A mi padre y mi

hermano, quienes a pesar de la distancia estuvieron siempre conmigo, gracias por el cariño,

apoyo y por el ánimo para seguir adelante. Los amo muchísimo, sin ustedes esto no hubiera

sido posible.

To all – vor allem – para todos

THANK YOU – DANKE SCHÖN – MUCHAS GRACIAS

Curriculum Vitae

Camila Hernández

152

9 Curriculum Vitae

Personal information Name: Camila José Hernández Frederick Date of birth: March 20th, 1984 Place of birth: Caracas, Venezuela

Education Oct 2008 - Oct 2011 Continuation of the Ph. D. studies at the Albert-Ludwigs-Universität

Freiburg. Supervisor: Dr. Susana Andrade. Co-supervisor: Prof. Dr. Oliver Einsle

Oct 2007 - Oct 2008 Ph. D. studies, Georg-August-Universität Göttingen. Supervisor: Dr. Susana Andrade, Co-supervisor: Prof. Dr. Oliver Einsle

Sep 2004 - Dec 2005 Diploma thesis "Phylogenetic divergence estimated with mtDNA molecular clock in vectors of Venezuelan Equine Encephalitis Virus: Culex (Melanoconion) taeniopus and Cx. (Mel) cedecei ( iptera: Culicidae)”. Universidad Central de Venezuela. Supervisor: Dr Juan Carlos Navarro

Sep 2000 - Dec 2005 Studies in Biology, Universidad Central de Venezuela. Caracas, Venezuela Sep 1994 - Jul 2000 High school studies, Colegio Antonio Ortega Ordoñez, San Antonio de los

Altos, Venezuela

Awards Apr 2008 - present DAAD Scholarship (Deutscher Akademischer Austausch Dienst/German

Academic Exchange Service) for Doctoral studies in Germany. Dec 2005 Graduation Special award: Honor Degree (Biology Graduates “First

position” 1 of 35). Facultad de Ciencias. Universidad Central de enezuela (UCV). Caracas-Venezuela.

Work experience Oct 2007-Apr 2008 Research scientist, Georg-August-Universität Göttingen/Department of

Molecular Structural Biology Dec 2005–Jul 2007 Research scientist, Universidad Central de Venezuela /Instituto de

Zoología Tropical, Laboratorio de Biología de Vectores Caracas, Venezuela Oct 2004–July 2007 Teaching assistant Universidad Central de Venezuela /Instituto de

Zoología Tropical Caracas, Venezuela

inauguraldissertation - FreiDok plus

Documents

Transcript of inauguraldissertation - FreiDok plus