Comprehensive evaluation of solution nuclear magnetic resonance spectroscopy sample preparation for...

Abstract The preparation of high quality samples is a

critical challenge for the structural characterization of

helical integral membrane proteins. Solving the struc-

tures of this diverse class of proteins by solution nu-

clear magnetic resonance spectroscopy (NMR)

requires that well-resolved 2D 1H/15N chemical shift

correlation spectra be obtained. Acquiring these

spectra demands the production of samples with high

levels of purity and excellent homogeneity throughout

the sample. In addition, high yields of isotopically en-

riched protein and efficient purification protocols are

required. We describe two robust sample preparation

methods for preparing high quality, homogeneous

samples of helical integral membrane proteins. These

sample preparation protocols have been combined

with screens for detergents and sample conditions

leading to the efficient production of samples suitable

for solution NMR spectroscopy. We have examined 18

helical integral membrane proteins, ranging in size

from approximately 9 kDa to 29 kDa with 1–4 trans-

membrane helices, originating from a number of bac-

terial and viral genomes. 2D 1H/15N chemical shift

correlation spectra acquired for each protein demon-

strate well-resolved resonances, and >90% detection of

the predicted resonances. These results indicate that

with proper sample preparation, high quality solution

NMR spectra of helical integral membrane proteins

can be obtained greatly enhancing the probability for

structural characterization of these important proteins.

Keywords HSQC Æ Integral Membrane Proteins ÆNMR Æ Sample Preparation Æ Structural Genomics ÆTROSY

Introduction

Membrane protein structural genomics faces numerous

challenges, the most significant of which is, arguably,

sample preparation, including preparing crystals for X-

ray diffraction, 2D crystals for cryo-electron micros-

copy, aligned samples for solid-state nuclear magnetic

resonance spectroscopy (NMR) or isotropic solutions

for solution NMR. Recently, several publications have

demonstrated that numerous membrane proteins can

be cloned and expressed in Escherichia coli [1–3]. Here

R. C. Page Æ J. D. Moore Æ M. Sharma Æ R. Chase ÆF. P. Gao Æ T. A. CrossDepartment of Chemistry and Biochemistry, Florida StateUniversity, Tallahassee, Florida 32306-4390, USA

H. B. Nguyen Æ R. C. Page Æ J. D. Moore Æ M. Sharma ÆR. Chase Æ F. P. Gao Æ T. A. CrossNational High Magnetic Field Laboratory, 1800 E. PaulDirac Drive, Tallahassee, Florida 32310, USA

H. B. Nguyen Æ T. A. Cross (&)Institute of Molecular Biophysics, Florida State University,Tallahassee, Florida 32306-4380, USAe-mail: [email protected]

C. K. Mobley Æ C. R. SandersDepartment of Biochemistry and Center for StructuralBiology, Vanderbilt University, Nashville, Tennessee 37232-8725, USA

L. Ma Æ F. D. SonnichsenDepartment of Physiology & Biophysics, and the ClevelandCenter for Structural Biology, Case Western ReserveUniversity, Cleveland, Ohio 44106, USA

S. Lee Æ S. C. Howell Æ S. J. OpellaDepartment of Chemistry and Biochemistry, Universityof California, San Diego, CA 92093-0307, USA

J Struct Func Genom (2006) 7:51–64

DOI 10.1007/s10969-006-9009-9

123

ORIGINAL PAPER

Comprehensive evaluation of solution nuclear magneticresonance spectroscopy sample preparation for helical integralmembrane proteins

Richard C. Page Æ Jacob D. Moore Æ Hau B. Nguyen Æ Mukesh Sharma ÆRose Chase Æ Fei Philip Gao Æ Charles K. Mobley Æ Charles R. Sanders ÆLiping Ma Æ Frank D. Sonnichsen Æ Sangwon Lee Æ Stanley C. Howell ÆStanley J. Opella Æ Timothy A. Cross

Received: 28 February 2006 / Accepted: 21 May 2006 / Published online: 19 July 2006� Springer Science+Business Media B.V. 2006

we demonstrate that many integral membrane proteins

can not only be expressed in rich media, but also in the

minimal media required for isotopic labeling. In addi-

tion, we show 1H/15N chemical shift correlation spectra

for 18 proteins with molecular weights ranging up to

29 kDa and 1–4 transmembrane (TM) helices that

represent excellent starting points for structure deter-

mination by solution NMR spectroscopy. These high

quality solution NMR spectra were obtained utilizing

two distinct, yet robust purification and sample prep-

aration protocols, ‘reconstitution’ and ‘detergent ex-

change’. We present a thorough analysis of the two

protocols and demonstrate the viability of each pro-

tocol for producing high quality samples of helical

integral membrane proteins for solution NMR struc-

ture determination efforts.

Membrane proteins are a functionally diverse class

of proteins representing 30% of protein encoding

genes [4]. Analyses of prokaryotic genomes have found

that proteins with molecular weights between 12 kDa

and 36 kDa occur most frequently [5] and that the

majority of integral membrane proteins (IMPs) are

helical [6]. This results in a large number of helical

IMPs that are well within the range of study by solution

NMR. Current estimates also suggest that membrane

proteins account for 60% of drug targets [7]. Despite

these facts, less than 100 of the 28,000 protein struc-

tures in the Protein Data Bank are unique IMPs [8, 9].

The striking disparity between the importance of these

proteins and the small amount of structural data

available reveals the inherent difficulty involved in

their structural characterization. It is clear that struc-

tural characterizations of helical IMPs require the

production of high quality samples composed of a

homogeneously folded and stable state solubilized in a

membrane mimetic that permits the recording of high

resolution spectra.

IMP expression and subcellular localization

To permit structural characterization, milligram quan-

tities of purified helical IMPs must be produced [10].

The scientific community has long regarded the

expression of IMPs as a significant bottleneck for

membrane protein structural genomics, yet recent ad-

vances are demonstrating that high expression levels

are becoming more routine [11]. Current approaches

have demonstrated that cloning and expressing mem-

brane proteins of relatively small molecular weight

with a small number of TM helices can be achieved

with great efficiency. The use of BL21(DE3) host cells

has been successful in many instances, however

C43(DE3) host cells have been proposed to be less

prone to the cytotoxic effects caused by the expression

of very large IMPs. For example, expression of CitS, a

47.5 kDa Na+-dependent citrate carrier with 11 TM

helices, was shown to increase by up to five fold in

comparison to expression in BL21(DE3) host cells [12].

However, in our experience BL21(DE3) cells have

performed comparably to cultures of C43(DE3) for the

proteins discussed herein.

While the purification of protein expressed in the

cell membrane is often favored by structural biologists

over expression in inclusion bodies as a source of

protein, the isolation of protein solely from cell mem-

branes is often hampered by extremely low yields. We

have observed that IMP overexpression into the cell

membrane is typically accompanied by expression of

the IMP into inclusion bodies. This is likely the result

of saturation of the secretory pathway due to protein

expression occurring at a faster rate than IMP insertion

into the membrane [11]. Until now reports on the

isolation and refolding of IMPs from inclusion bodies

have been limited to a small number of proteins [13–

15]. Our results for the proteins in this report show

remarkable success in isolating and refolding proteins

from inclusion bodies in quantities sufficient for

structural characterization by solution NMR.

Important factors in IMP purification

Robust protocols are necessary for the isolation and

purification of protein to produce homogeneous IMP

preparations. The detergent mediated isolation of

IMPs is routinely achievable and has been discussed in

detail elsewhere [16] as has purification via Ni2+ affinity

chromatography for efficiently producing protein at

purity levels acceptable for characterization by NMR

[17]. The level of purity needed for each protein is

dependent upon the identity of the contaminants. The

presence of contaminating lipids and low molecular

weight proteins can introduce unwanted heterogeneity

into the sample, broadening linewidths or even intro-

ducing spurious resonances. This was clearly illustrated

by the detection of the E. coli protein YodA in some

IMP samples purified by Ni2+affinity chromatography

from E. coli membrane fractions [17]. Low levels of

high molecular weight protein contaminants, while

undesirable, may be tolerated if they do not interact

with the protein of interest since the low concentration

and long rotational correlation times of these proteins

typically prevent them from being observed by

NMR. However, even very low concentrations of

low molecular weight contaminants can prove trou-

blesome. Non-proteinaceous contaminants can also

result in increased sample heterogeneity. Non-essential

52 J Struct Func Genom (2006) 7:51–64

123

lipid–protein interactions may allow host cell lipids to

remain bound to the protein throughout the Ni2+

affinity purification. In these cases further purification

via HPLC may be necessary to effectively remove the

contaminants and improve sample homogeneity.

Choosing the membrane mimetic: considerations

and limitations

An ideal membrane mimetic for solution NMR will

solubilize the IMP without loss of protein activity and

allow for the acquisition of well-resolved spectra. Fur-

thermore the protein should be stable enough to permit

a series of multidimensional NMR experiments to be

acquired on a single sample. Detergent micelles can

satisfy these requirements and provide good models of

membrane interfaces for small proteins [18]. Recent

literature suggests a number of choices for detergents

that allow high-resolution data to be acquired while

retaining protein function [19]. These choices are nec-

essary since different detergents have been essential for

solubilizing the few helical IMPs that have been studied

(e.g. SDS for MerF [20], DHPC for Vpu [21] and DPC

for phospholamban [22, 23]). For example, the lyso-

phosphatidylglycerol (lyso-PG) detergents are widely

applicable, yet these detergents have some stability

problems [24]. Additionally, different proteins with

varied amino acid compositions and topologies will

exhibit a wide range of interactions between the pro-

tein, detergent headgroups, and acyl chains. Unfortu-

nately the best detergent for a particular protein cannot

be determined a priori, rather it must be determined by

screening a number of detergents and sample condi-

tions. Similarly, screens have been established to

determine crystallization conditions. Here we demon-

strate that with proper sample preparation, highly re-

solved spectra can be obtained for a range of proteins in

a number of different detergents.

Solution NMR characterization of helical IMPs

While X-ray crystallography has provided the majority

of protein structures to date, significant challenges

have been encountered in the realm of membrane

proteins. Notably, only 20% of proteins purified by

structural genomics efforts have resulted in diffraction

quality crystals [25]. Solution NMR is an attractive

method to structurally characterize many helical IMPs,

yet few examples of the high quality spectra necessary

for a structural effort have been achieved. For helical

IMPs, spectral resolution is complicated by the limited

amide 1H chemical shift dispersion in a-helices and the

slow correlation time for many micelle bound proteins.

The feasibility of solution NMR based structural

characterization can be examined using the two-

dimensional 1H/15N-heteronuclear single-quantum

coherence (HSQC) experiment [26]. These spectra

correlate amide proton and nitrogen pairs within the

protein and serve as a building block for a multitude of

experiments that yield the three- and four-dimensional

triple resonance spectra essential for sequential back-

bone resonance assignment. Obtaining a sufficiently

resolved 1H/15N-HSQC spectrum is a prerequisite for

NMR based structural characterization. To enhance

resolution, TROSY based pulse sequences have been

applied to reduce resonance linewidths for some

membrane proteins [27]. The single most critical step,

however, in obtaining these high quality spectra is in

developing a sample preparation protocol that effi-

ciently and reliably produces homogeneous samples.

Idealized properties of an IMP solution NMR

sample

For this report we define high quality samples as those

capable of producing high-resolution solution NMR

spectra sufficient for structure determination. High

quality samples for solution NMR can be primarily

characterized by two parameters, sample purity and

homogeneity. Uniform labeling with stable isotopes

results not only in the labeling of the protein of inter-

est, but also every other molecule within the cell.

Proper purification is essential to limit the number of

resonances observed to only those originating from the

protein of interest. The second parameter, sample

homogeneity, is much more challenging to achieve.

The complexity of creating a homogeneous sample is a

direct consequence of the number of weak molecular

interactions the protein has with its heterogeneous

environment. Structurally inhomogeneous samples

give rise to spectra with either a higher number of

observed resonances than predicted, or a dramatic in-

crease in resonance linewidths and a reduction in the

ability to resolve individual resonances. The observa-

tion of more resonances than expected occurs when

there are multiple protein conformations and when

there are multiple populations of micelles within a

sample. Dramatic increases in resonance linewidths are

caused by a myriad of interactions and dynamics. A

high quality NMR sample therefore has high purity,

high structural homogeneity and an environment that

is the same for each protein. We herein report recent

progress in the sample preparation of helical IMPs for

solution NMR spectroscopy that represents significant

progress towards accelerating the rate at which mem-

brane protein structures can be solved.

J Struct Func Genom (2006) 7:51–64 53

123

Materials and methods

Expression of helical IMPs

Fourteen helical IMPs (Rv0008c, Rv0820, Rv0985c,

Rv1031, Rv1567c, Rv1616, Rv1761c, Rv1861, Rv2433c,

Rv2719c, Rv3004, Rv3069, Rv3368, Rv3773c) identi-

fied from the Mycobacterium tuberculosis genome [28]

were cloned into modified pET-16b or modified

pET-29b vectors producing fusion constructs with

either N- or C-terminal His6 tags respectively [1].

Proteins from M. tuberculosis were expressed in E. coli

BL21(DE3)CodonPlus-RP cells at levels ranging from

approximately 1–100 mg/l of minimal media (Table 1).

Proteins from the M. tuberculosis genome were chosen

based on molecular weight and number of transmem-

brane helices from an expression library of 143

M. tuberculosis integral membrane proteins. The M2

proton channel from Influenza A virus was cloned and

expressed at 100 mg/l miminal media as described

previously [29]. The proteins KcsA, MerFm and Vpu

were cloned and expressed as previously reported [20,

29, 30]. The number of TM helices reported for each

protein were either predicted by TMHMM v2.0 or

determined from experiments in previous literature

[31, 32].

Purification for reconstituted and detergent

exchanged samples

After cell lysis helical IMPs were purified by one of

two methods, either ‘reconstitution’ or ‘detergent

exchange’. A brief overview of the two sample

preparation protocols is given by the flow chart in

Fig. 1. Protein purified via the ‘reconstitution’

method was solubilized either from cell membranes

or under denaturing conditions from inclusion bodies.

Solubilized protein from both inclusion bodies and

membrane fractions were purified separately with

Ni2+ affinity chromatography. Chromatographic

refolding was utilized to simultaneously refold and

purify protein isolated from inclusion bodies [13].

Purified protein was extensively dialyzed to remove

detergent and subsequently lyophilized for later

sample preparation steps. Samples purified via the

‘detergent exchange’ method were isolated from cell

membrane fractions under mildly denaturing condi-

tions. Proteins were refolded and purified by Ni2+

affinity chromatography and on-column detergent

exchange [17, 33]. Purified protein was eluted in the

desired detergent for subsequent NMR spectroscopy.

In Fig. 2 the purity of 3 helical IMPs is shown by

SDS PAGE chromatography.

Frozen or freshly harvested cells to be lysed were

resuspended in 20 ml lysis buffer per gram of wet cells.

Lysis buffer for the ‘reconstitution’ protocol consisted

of 20 mM Tris–HCl, 500 mM NaCl, pH 7.9 and for the

‘detergent exchange’ protocol it consisted of 75 mM

Tris–HCl, 300 mM NaCl, 0.2 mM ethylenediamine-

tetraacetic acid (EDTA), pH 7.7. Phenylmethylsul-

fonyl fluoride, magnesium acetate, lysozyme (Sigma)

and either Benzonase (Novagen) or DNase and RNase

were added to both lysis buffers at final concentrations

of 1.1 mM, 5 mM, 0.2 mg/ml and 0.02 mg/ml, respec-

tively. Cells for both protocols were incubated at room

temperature for 30 min on a rotary shaker followed by

sonication in an ice bath for 5 min at 50% duty cycle

(repetitions of 5 s on, 5 s off).

For ‘reconstituted’ samples, inclusion bodies were

isolated from crude lysate via centrifugation at

15,000· g for 20 min. Inclusion bodies were solubilized

in 6 M urea and 0.5% Empigen (Calbiochem) in Buffer

A (20 mM Tris–HCl pH 7.9, 500 mM NaCl, 5 mM

imidazole). Solubilized inclusion bodies isolated from

1 l of culture were loaded onto 20 ml Ni-NTA resin

(Qiagen) equilibrated with the same buffer. Bound

protein was refolded by removal of urea in 1 M

increments using 2 column volumes per increment until

all urea was removed, followed by thorough washing

with Buffer A containing 0.5% DPC (dodecylphosph-

ocholine) and 40 mM imidazole. Protein eluted in

Buffer A with 400mM imidazole and 0.5% DPC was

thoroughly dialyzed against distilled deionized H2O

using 8,000 Da molecular weight cutoff regenerated

cellulose dialysis tubing (Fisher) for 48 h with six buf-

fer changes to remove detergent and to precipitate the

protein. Precipitated protein was repeatedly resus-

pended, washed, and centrifuged in 50:50 acetone:H2O

to remove contaminating detergents and lipids, and

subsequently lyophilized. The protein was stored at –

80�C until used for NMR sample preparation. Further

purification to remove additional contaminants was

conducted for M2, KcsA, MerFm and Vpu proteins via

HPLC as previously described [20, 29, 30].

Following removal of insoluble components from

crude lysates by centrifugation at 15,000· g for 20 min,

cell membranes were isolated from the remaining

supernatant via ultracentrifugation at 90,000· g for 3 h.

IMPs for ‘reconstituted’ samples were extracted from

cell membranes by resuspension in Buffer A with 0.5%

Empigen followed by incubation for 2 h at room tem-

perature on a rotary shaker. Insoluble components were

removed by ultracentrifugation at 90,000· g for 3 h. The

supernatant containing extracted IMPs was loaded onto

Ni-NTA resin equilibrated with Buffer A with 0.5%

Empigen. Bound protein was washed thoroughly with


123

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123

Buffer A containing 0.5% DPC and 40 mM imidazole.

The protein was eluted, precipitated, washed and stored

as describe above for protein from inclusion bodies.

The purification of protein for ‘detergent exchanged’

samples was performed as described previously [17, 33].

Briefly, Empigen was added to crude lysates to a final

concentration of 3% and incubated at 4�C with tum-

bling for 30 min. Extracted lysate was centrifuged at

18,000· g for 20 min to remove insoluble components.

The supernatant was added to Ni-NTA resin (1.2 ml per

Fig. 1 Helical IMP solution NMR samples were prepared viaeither the ‘detergent exchange’ or ‘reconstitution’ methods. Theflow chart illustrates the key differences between the protocols in

terms of protein source, solubilization, purification, and finalsample preparation steps


123

gram wet cells) equilibrated with Buffer B (40 mM

HEPES, 300 mM NaCl, 10 mM butylated hydroxytol-

uene, pH 7.5) and incubated on a rotary shaker for

30 min at 4�C before being transferred to an empty

column. Resin with bound protein was washed with 3

column volumes of ice-cold Buffer B with 3% Empigen

followed with an additional 3 column volumes of Buffer

B with 3% Empigen, 40 mM imidazole and 0.5 mM

DTT. Rinsing the resin with 8 column volumes of cold

rinse buffer (25 mM sodium phosphate, 0.5% DPC,

pH 7.2) completed the detergent exchange process. The

protein was eluted in 250 mM imidazole, pH 7.2, 0.5%

DPC. Since the detergent used in the rinse and elution

steps will be the detergent present in the final NMR

sample, the 0.5% DPC may be substituted with 0.5%

LMPG (1-myristoyl-2-hydroxy-sn-glycero-3-[phospho-

RAC-(1-glycerol), 0.5% LPPG (1-palmitoyl-2-hydro-

xy-sn-glycero-3-[phospho-RAC-(1-glycerol), 0.2% SDS

(sodium dodecyl sulfate) or 0.2% Sarcosyl (sodium N-

laurylsarcosinate) during these steps. Purification via

nickel affinity chromatography for both ‘reconstitution’

and ‘detergent exchange’ methods typically resulted in

samples with greater than 95% purity.

Helical IMP sample preparation via reconstitution

or detergent exchange

‘Reconstituted’ samples were prepared by dissolving

lyophilized protein in approximately 400 ll 5% w/v

detergent with 90:10 H2O:D2O via repeated cycles of

heat (45�C) and bath sonication. These samples were

centrifuged to remove debris, and pH was adjusted

with acetic acid before being dispensed into a 5 mm i.d.

Shigemi NMR tube (Allison Park, PA). For ‘detergent

exchanged’ samples, D2O was added to the eluted

protein to a final concentration of 10%. To chelate free

nickel ions, EDTA was added to a final concentration

of 0.5 mM. Sample pH was adjusted with perdeuter-

ated acetic acid. Each sample was concentrated to a

final volume of approximately 600 ll using Millipore

Amicon Ultra 5 kDa cutoff centrifugal filters (Bille-

rica, MA) before being dispensed into a 5 mm i.d.

Wilmad (Buena, NJ) 528-PP or better thin walled

NMR tube.

NMR spectroscopy of helical IMP samples

1H/15N-HSQC spectra were acquired for 18 proteins

at proton frequencies ranging from 500 MHz to

900 MHz. For each protein a number of conditions

were tested including type of detergent, detergent

concentration, buffer pH, and sample temperature. For

the sake of brevity, only those spectra and corre-

sponding conditions giving rise to the highest quality1H/15N-HSQC spectra for each protein are shown.

Detailed descriptions of the conditions used for each

protein are located in Table 1. Spectra were typically

acquired on samples with protein concentrations

ranging from 1 mM to 1.5 mM. Datasets designated as

HSQC were acquired utilizing a gradient-enhanced

HSQC pulse sequence [34]. Datasets designated as

TROSY were acquired utilizing a spin-state selective

gradient-enhanced TROSY pulse sequence [17, 35].

Spectra were processed using NMRPipe [36] and

figures generated with Sparky [37].

Table 2 Average 1H and 15N linewidths and number of observedamide backbone resonances (as percent of predicted resonances)within ‘reconstituted’ and ‘detergent exchanged’ samples for

1H/15N-TROSY spectra of Rv0008c in 250 mM SDS, pH 4.15 at50�C and 1H/15N-HSQC spectra of KdpC (Rv1031) in 150 mMDPC, pH 4.1 at 50�C

Rv0008c KdpC (Rv1031)

RCa DEb RC DE

Average 1H dimension 23.3 25.0 25.3 19.8Linewidth (Hz) 15N dimension 18.5 18.8 18.9 15.6Percent observed peaks 90% 90% 94% 79%

a Sample prepared via reconstitution into detergent as discussed in Materials and Methodsb Sample prepared via detergent exchange as discussed in Materials and Methods

Fig. 2 Proteins presented in this report were typically purified togreater than 95% purity. Purified samples of the proteinsRv0008c, MscL (Rv0985c), and KdpC (Rv1031) were analyzedusing a 12% polyacrylamide gel with SDS. Protein bands werevisualized by Coomassie Brilliant Blue staining


123

Results and discussion

Protein expression for NMR spectroscopy

Utilizing E. coli BL21(DE3) cells we have achieved

robust expression levels up to 100 mg per liter of

minimal media for uniform 15N labeling as illustrated

in Table 1. Since a reduction in expression yield is

expected when culturing cells in minimal media, stable

expression platforms are paramount [38]. The com-

plete replacement of spent media and resuspension

into fresh isotope labeled media prior to induction has

been demonstrated to increase yields [39]. We have

further enhanced expression levels in minimal media

by increasing cell densities through a 4:1 concentrating

method [40]. High cell densities were therefore

achieved by growing 4 l of cell culture to O.D.600~0.7 in

LB media followed by centrifugation at 8,000· g for

8 min, resuspension into 1 l M9 minimal media, and

subsequent induction of expression after a short

equilibration time. Higher expression levels were

attained by effectively increasing the cell mass per unit

volume, reducing consumption of stable isotope

labeled compounds, and allowing expression to take

place during log phase growth.

The expression of large quantities of protein often

results in the formation of inclusion bodies and there-

fore, we frequently observe the expression of helical

IMPs in inclusion bodies, while high expression into

the cell membrane is relegated to a small number of

proteins [1]. Of the 18 proteins presented in this report,

only 5 resulted in quantifiable levels of expression in

the cell membrane. All five of these proteins exhibited

concomitant expression into inclusion bodies. Those

15N

Che

mic

al S

hift

(pp

m)

1H Chemical Shift (ppm)

c106

107

108

109

110

111

8.6 8.4 8.2 8.0

a105

110

115

120

125

7.8 7.68.6 8.4 8.2 8.08.8

d

** *

8.6 8.4 8.2 8.0

b

7.8 7.68.6 8.4 8.2 8.08.8

*

Fig. 3 1H/15N-TROSYspectra are nearly identicalfor Rv0008c in 150 mM DPC,pH 4.15 at 50�C prepared via(a) ‘reconstitution’ and (b)‘detergent exchange’ samplepreparation methods.1H/15N-HSQC glycineresonances of KdpC (Rv1031)in 150 mM DPC, pH 4.1 at50�C indicate differingnumbers of resonances forsamples produced by (c)reconstitution or (d)detergent exchange. Asterisksdenote locations of missingresonances present in 2c butabsent in 2d


123

cases exhibiting high expression levels in the cell

membrane are likely attributed to the production of

highly invaginated or other proliferated membrane

structures produced by the host cell in response to IMP

overexpression [41].

Sample preparation via ‘reconstitution’

or ‘detergent exchange’

Two primary methods for sample purification were used

in this study: ‘reconstitution’ and ‘detergent exchange’

as discussed in Materials and Methods. Here, solution

NMR samples of Rv0008c and KdpC (Rv1031) were

prepared via these two distinct methods. Both methods

produced nearly identical high quality samples. The

Rv0008c sample prepared by ‘reconstitution’ utilized

protein isolated from inclusion bodies, while the sample

prepared by ‘detergent exchange’ used protein isolated

from the cell membrane. Samples of KdpC (Rv1031)

prepared by ‘reconstitution’ and ‘detergent exchange’

each utilized protein isolated from inclusion bodies.

Spectra obtained via ‘reconstitution’ and ‘detergent

exchange’ for Rv0008c suggest similar well-folded

homogeneous samples from the two protocols (Fig. 3a,

b and Table 2). The number of observed peaks was

invariant between the two methods for this protein and

the difference in linewidths between the two methods is

less than 1 Hz in the 15N dimension, and 1.7 Hz in the 1H

dimension. ‘Detergent exchange’ and ‘reconstitution’

are both options for the production of helical IMP NMR

* e

g

b c

d f

h * i

125

120

115

110

105

125

120

115

110

105

125

120

115

110

105

9.0 8.5 8.0 7.5 7.0 9.0 8.5 8.0 7.5 7.0 9.0 8.5 8.0 7.5 7.0

* a

15N

Che

mic

al S

hift

(pp

m)

1H Chemical Shift (ppm)

Fig. 4 The effect of differentmicellar environments uponthree different proteinsillustrated with 1H/15N-HSQCspectra. The detergentsLMPG (a, d, g), DPC (b, e, h)and SDS (c, f, i) were used toobtain spectra of Rv2433c (a–c), Rv1761c (d–f) and MscL(Rv0985c) (g–i). Asterisksdenote best spectra for eachprotein based on linewidthsand percent of predictedresonances observed. Spectrafor Rv1761c and Rv2433cwere acquired at pH 4 and50�C. Spectra for Rv0985cwere acquired at pH 3.7 and50�C


123

samples. However, the results for KdpC (Rv1031)

illustrated in Fig. 3c, d and Table 2 demonstrate that

while both sample preparation protocols are capable of

producing samples of high quality, the results are not

always the same. While the linewidths for the ‘detergent

exchanged’ sample are slightly narrower compared to

the ‘reconstituted’ sample, the number of peaks ob-

served in the ‘detergent exchanged’ sample of KdpC

(Rv1031) is 15% lower than for the ‘reconstituted’

sample. In addition, significant chemical shift differ-

ences are seen for the observed resonances suggesting

an altered conformation. Without resonance assign-

ment, or a three-dimensional structure it is not possible

to pinpoint the reason for the loss in number of reso-

nances. Despite the differences in the two spectra for

KdpC, the results are highly reproducible from sample

to sample demonstrating that both ‘reconstitution’ and

‘detergent exchange’ are capable of generating repro-

ducible homogeneous samples that give rise to high-

resolution spectra. Different conformation/dynamic

states are a cause for concern, but not as much as it

would be for water-soluble proteins where multiple

conformational states are a rarity. Membrane proteins

are known to have multiple conformational states and it

is possible that both of these states bare a resemblance to

a native state for this protein.

Variations in spectral quality with different

detergents

Since it is not currently possible to determine the best

detergent a priori, a number of different detergent

types including anionic (SDS and Sarcosyl), long chain

(LMPG and LPPG), medium chain (DPC), and short

chain (DHPC) zwitterionic detergents were screened

before obtaining the solution NMR spectra shown

herein. Detergent screens via analysis of a series of1H/15N-HSQC spectra are vital for selecting the

appropriate detergent for each protein. As shown in

Fig. 4, the quality of heteronuclear NMR spectra varies

greatly as a function of detergent [19, 24, 42, 43]. It is

not surprising that different detergents have such dra-

matic effects upon the spectra, as a number of complex

interactions are involved. Furthermore the shape and

dynamics of the detergent micelles can vary with pH

and temperature thereby altering the environment

surrounding the IMP [44]. While it is hoped that

samples giving rise to such high-resolution spectra

reflect a native-like state, it was not the primary con-

cern of this study to check for functionality. In fact, for

13 of the proteins in this report, the function is un-

known and cannot be reliably assayed. The degree to

which the membrane mimetic environment influences

the structure of a protein remains a question. Indeed,

we would argue that all stable, homogeneous structures

are of interest and provide a description of the low

energy structural landscape for a given protein.

Moreover, since many proteins have multiple func-

tional states one would need multiple functional assays

to identify these functional states.

It is common that a detergent may perform very well

under one set of conditions, yet poorly at a different

pH or temperature. Ideally the detergent micelles will

interact with the helical IMPs to sufficiently solubilize

the protein without disrupting the packing of TM

helices or distorting regions of the protein outside the

membrane. The spectra shown in Fig. 4 illustrate the

performance of the detergents LMPG, DPC and SDS

with the proteins Rv2433c, MscL (Rv0985c) and

Rv1761c. While for this set of proteins a different

detergent is optimal for each protein, DPC appears to

be the detergent most frequently found to form the

high-resolution samples (Table 1). The spectroscopic

goal for these helical IMPs was to achieve a stable

conformation that is homogeneous throughout the

sample resulting in spectra with narrow linewidths and

the expected number of observed resonances (Fig. 4a,

e, i). Spectra with broad linewidths and missing reso-

nances (Fig. 4b) are indicative of a micellar environ-

ment for which the protein is aggregated or has slow

global motions resulting in poor spectral properties

throughout the sample. Alternatively, the presence of

more resonances than expected may indicate the

presence of multiple conformations of the IMP or

variations among the detergent micelles within the

sample.

The varying performance of detergents is under-

scored by the range of detergents that have been used

for structural characterization of helical IMPs. The

structures of MerF [20], Vpu [21], and phospholamban

[22, 23] were solved in SDS, DHPC and DPC respec-

tively. These detergents are capable of forming sam-

ples that are stable for extended periods of time. The

lyso-PG detergents LMPG and LPPG have been pro-

posed as superior membrane mimetics [24], yet no

structures of helical IMPs have been reported in lyso-

PG detergents. While the lyso-PG detergents can

produce very high quality spectra, we have found that

proteins solubilized in these detergents exhibit poor

sample stability. Typically we find that lyso-PG sam-

ples are stable only for several days at room temper-

ature, and extension of sample lifetimes is only possible

by storing samples at elevated temperatures (40–50�C).

Unfortunately storing samples at elevated tempera-

tures increases the risk for proteolysis and sample

degradation. The stability of helical IMP sample


123

preparations is important since a number of multidi-

mensional spectra must be obtained for structural

characterization, making sample lifetimes longer than

one week highly desirable.

Uniformity of resonance intensities within spectra

The variation of resonance intensities within each

spectrum can be a function of the detergent. These

variations are likely a result of differing dynamics in

spatially disparate regions of the protein or a result of

sample heterogeneity. For instance, spectra of Rv0985c

(Fig. 4g, h, i) differ significantly in DPC and SDS. In

Fig. 5 the number of weak resonances in any given

spectrum is small although they are more pronounced

in some spectra, such as that for Rv3069, yet very

uniform in other spectra, such as that for MerFm. In

light of this it may not be surprising that a structure has

been solved by solution NMR for MerFt [20]. How-

ever, simply the presence of dissimilar resonance

intensities does not negate the possibility that a struc-

ture may be solved, and does not indicate that the

structure is non-native. For example, the spectrum of

Vpu contains resonances with varied intensities yet a

structure was solved utilizing solution NMR [21]. It

remains to be seen quantitatively how much variation

in resonance intensities is allowable for structural

characterization of these proteins. Yet it is promising

that even with significant resonance intensity varia-

tions, structures are still attainable.

125

120

115

110

105

1 H Chemical Shift (ppm)

9.0 8.5 8.0 7.5 7.0 9.0 8.5 8.0 7.5 7.0 9.0 8.5 8.0 7.5 7.0

125

120

115

110

105

125

120

115

110

105

15N

Che

mic

al S

hift

(pp

m)

Rv0008c

SDS

1/18.4

RC

90

T

PhoT(Rv0820)

DPC

1/29.2

DE

99

T

KdpC (Rv1031)

DPC

1/21.1

RC

94

H

Vpu

DHPC

1/9.0

RC

100

H

M2

LPPG

1/11.9

RC

99

T

Rv2719c

DPC

1/18.4

RC

92

H

Rv1761c

DPC

1/16.2

RC

95

H

Rv3004

LMPG

1/13.3

RC

94

H

Rv3368

Sarcosyl

1/24.8

DE

90

T

Fig. 5 High-resolution15N/1H chemical shiftcorrelation spectra for 18helical integral membraneproteins have been obtainedwith an average of 95%detection of predictedresonances. Spectra areorganized by number of TMhelices and percent-observedresonances. Figure insetsdenote the protein name and/or ORF number anddetergent used. The numberof transmembrane helices andmolecular weight (kDa) areseparated by a backslash. Thesample preparation method isdenoted as either RC(reconstitution) or DE(detergent exchange). Percentobserved resonances iscalculated as (# observedresonances/# predictedresonances) · 100%. Spectrawere obtained utilizing eithergradient enhanced HSQC (H)or TROSY (T) pulsesequences. Acquisitionparameters and sampleconditions for all 18 proteinsare shown in Table 1


123

Advances in helical IMP spectral quality

Obtaining high quality two-dimensional 1H/15N corre-

lation spectra is imperative for structural character-

ization by solution NMR. The quality of two-

dimensional spectra obtained by HSQC and TROSY

pulse sequences are generally similar for moderate-

sized helical IMPs in detergent micelles. We have ob-

tained high quality HSQC and TROSY spectra for 18

IMPs having molecular weights between 9 and 29 kDa

and 1 and 4 TM helices as shown in Fig. 5. A wide

variation in the performance of five commonly used

detergents was seen. It is clear that high quality spectra

can be obtained in DPC, SDS, Sarcosyl, DHPC, LMPG

and LPPG over a range of molecular weights and

topologies for helical IMPs as illustrated in Fig. 5 and

Table 1. DPC performs well in many cases, solubilizing

nearly all of the proteins examined and giving excellent

quality spectra in a number of cases. The lyso-PG

detergents produced very high quality spectra for 4

proteins that gave poor results in DPC. SDS was found

to produce highly resolved spectra for a number of

proteins.

The samples and corresponding solution NMR

spectra reported herein represent excellent prospects

for structure determination not unlike the prospects for

structural characterization once a well diffracting

crystal is obtained. The structural characterization of

proteins via solution NMR spectroscopy requires a

number of multidimensional experiments for backbone

resonance assignment and collection of distance and

orientational restraints [46, 47]. The 1H/15N-HSQC

125

120

115

110

105

1 H Chemical Shift (ppm)

9.0 8.5 8.0 7.5 7.0 9.0 8.5 8.0 7.5 7.0 9.0 8.5 8.0 7.5 7.0

125

120

115

110

105

125

120

115

110

105

15N

Che

mic

al S

hift

(pp

m)

Rv3773c

DPC

2/22.3

DE

93

T

MscL (Rv0985c)

SDS

2/17.2

RC

96

H

Rv2433c

LMPG

2/11.3

RC

98

H

Rv1567c

LMPG

2/11.6

RC

92

T

MerFm

SDS

2/8.7

RC

100

H

Rv1861

SDS

3/11.3

RC

93

H

KcsA

SDS

2/11.0

RC

90

H

Rv3069

DPC

4/15.4

DE

98

T

Rv1616

Sarcosyl

3/15.3

DE

96

T

Fig. 5 continued


123

experiment serves as a tool to predict the possible

success or failure of a sample for these more complex

experiments. To offer the best chance for success, one

must obtain a 1H/15N-HSQC spectrum that resolves as

many resonances as possible; typically 90% or more is

desired. Without this base level of spectral quality the

amount of information available in latter experiments

will be severely compromised, limiting the possibility

for successful structure determination. Each of the 18

spectra illustrated in Fig. 5 exhibit both the resolution

and percent detection of resonances needed to justify

further experiments for structural characterization. In

fact the 1H/15N-HSQC spectra in Fig. 5 for MerF and

Vpu have permitted the structural characterization of

these proteins [20, 21].

Conclusions

There have been numerous challenges in the struc-

tural characterization of helical IMPs by solution

NMR, but similar to other methodologies the most

critical challenge is sample preparation. To accom-

plish this goal a dependable expression system had to

be established that would provide adequate quantities

of isotope labeled protein. Robust purification

protocols, a streamlined detergent screen and

optimized sample conditions were developed leading

to an increased probability of success for obtaining

high-resolution spectra. The resulting samples are

stable for days to weeks and each represents a

homogeneous preparation in a folded conformation.

All of these hurdles have been overcome in demon-

strating numerous high quality spectra that provide

excellent prospects for structural characterization of

helical IMPs.

The expression systems employed here allow for

high expression levels into a combination of inclusion

bodies and cell membranes. Both the ‘reconstitution’

and ‘detergent exchange’ sample preparation methods

are fully capable of producing homogeneous prepara-

tions of protein extracted from inclusion bodies or cell

membranes. While these methods are capable of pro-

ducing high quality samples, a wide range in spectral

quality can be seen with different conditions, the most

dramatic of which is associated with the choice of

detergent. Although changes in sample conditions

yield variations in spectral quality, we have shown that

the appropriate conditions can often be found for each

protein that gives rise to high-resolution solution NMR

spectra. Furthermore, we have defined conditions that

represent a high probability starting point for structural

characterization.

The quality of spectra now attainable for helical

integral membrane proteins indicates that we have

achieved efficient and robust sample preparation pro-

tocols applicable to a range of proteins. These spectra,

obtained for proteins ranging in size from approxi-

mately 9–29 kDa with 1–4 TM helices, demonstrate

well-resolved resonances and an average of 95%

detection of the predicted resonances. While these

spectra do not represent a guarantee that a three-

dimensional structure can be determined, in much the

same way that a well diffracting crystal does not

guarantee a structure, these spectra do represent suc-

cess for crossing the most challenging hurdle in mem-

brane protein structural biology; that of sample

preparation. These advances in helical IMP sample

production for solution NMR spectroscopy will greatly

improve the speed with which structures of these pro-

teins may be solved, opening the doors to a realm of

proteins for which very little is known.

Acknowledgements The authors thank T.M. Logan of FloridaState University for his helpful discussion concerning initialsolution NMR spectroscopy of helical IMPs from M. tuberculo-sis. The authors also thank B. Xu, D.H. Jones, and S.H. Park ofUniversity of California, San Diego, and H. Qin and Y. Hua ofFlorida State University for their contributions to the work. Thiswork was supported by NIH grant PO1 GM064676 and NSFgrant MCB-0235774. A portion of this work was performed atthe National High Magnetic Field Laboratory funded by theNational Science Foundation (DMR 0084173) and the State ofFlorida. Parts of this research were performed in the Environ-mental Molecular Sciences Laboratory (a national scientific userfacility sponsored by the U.S. DOE Office of Biological andEnvironmental Research) located at Pacific Northwest NationalLaboratory, operated by Battelle for the DOE. This researchbenefited from activities at the Southeast Collaboratory forHigh-Field Biomolecular NMR, a research resource at theUniversity of Georgia, funded by the National Institute ofGeneral Medical Sciences (NIGMS grant number P41GM066340) and the Georgia Research Alliance. The researchutilized the Biomedical Technology Resource for NMR Molec-ular Imaging of Proteins supported by NIH grant P41 EB002031.C.K. Mobley was supported by NIH training grantT32GM008320. J.D. Moore was supported by NIH predoctoralfellowship F31NS054494. R.C. Page and H.B. Nguyen weresupported by American Heart Association predoctoral fellow-ships.

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