biomimetic membranes as new materials for - CORE

BIOMIMETIC MEMBRANES AS NEW MATERIALS FOR APPLICATIONS IN ENVIRONMENTAL ENGINEERING AND BIOLOGY

BY

MANISH KUMAR

DISSERTATION

Submitted in partial fulfillment of the requirements

for the degree of Doctor of Philosophy in Environmental Engineering in Civil Engineering in the Graduate College of the

University of Illinois at Urbana-Champaign, 2010

Urbana, Illinois

Doctoral Committee:

Research Assistant Professor Julie L. Zilles, Chair Professor Mark M. Clark, Director of Dissertation Research Emeritus Professor Vernon L. Snoeyink Professor Wolfgang P. Meier, University of Basel

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ABSTRACT

Biological water channel proteins, called aquaporins, provide selective and rapid transport of

water across cell membranes. They utilize an elegant mechanism distinct from and more

efficient than that used in commercial solute separation polymeric membranes such as Reverse

Osmosis (RO) membranes. In this work, the bacterial Aquaporin (AqpZ) was functionally

incorporated into synthetic biomimetic polymer vesicles. Using stopped flow light scattering, the

permeability of such systems was determined to be up to two orders of magnitude higher than

current RO membranes, revealing the potential of this approach. A templating procedure was

then used to make flat membrane films with a high density of AqpZ. The sizes of these films are

small (~500 nm) and more research needs to be performed to scale up this process. However,

this method led to creation of flat 2D or thin 3D crystals of AqpZ in a polymer matrix as

confirmed by electron diffraction. This indicates that the packing efficiency of these polymer-

based systems is extremely high. Additionally, such crystals have the potential to allow for

structural reconstruction of the incorporated aquaporins. This procedure can thus provide

fundamental knowledge regarding the conformation of membrane proteins in block copolymers

and help in design of functional protein-polymer hybrid materials.

This work also led to the serendipitous discovery of AqpZ gating (reversible closure) at low pH

values when incorporated into triblock copolymer vesicles. This gating is also present in bacteria

and has relevance for bacterial survival under acid and osmotic shock. An overall scheme of

osmoregulation and acidic shock survival utilizing coordinated activation and gating of

membrane proteins is proposed.

Several research ideas resulted from this work and are currently being pursued. This includes

determination of insertion efficiency of membrane proteins in block copolymers, the use of block

copolymer membranes for studying gas transport in membrane proteins, block copolymer

vesicles with encapsulated perchlorate degrading enzymes for water treatment, and carbon

capture using active CO2 transporters inserted into block copolymer membranes.

Overall, this work has demonstrated the promise of using hybrid protein-polymer systems for

environmental engineering applications. In particular its applicability to synthetic desalination

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membranes is most promising and relevant. The basic approach used here may be applied to any

separation for which a specific transport protein is available or could be engineered. My work

has also contributed to understanding the properties of aquaporins, in particular AqpZ and its

possible role in microbial physiology. Finally, recent successes in immobilizing protein

molecules and in synthesizing 2D crystals of membrane proteins may provide an excellent way

to answer fundamental questions regarding the structure and function of these membrane

proteins in block copolymers.

In broad terms, this work has shown that biology provides excellent paradigms for engineering

materials and processes that are efficient and sustainable and that this “reverse engineering”

approach can enrich our understanding of underlying biological phenomena

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To Anu, without whom all this would not be possible.

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ACKNOWLEDGMENTS

This work needed to be collaborative to achieve whatever modest level of success it has

achieved and I have many people to thank. First, I would like to thank my advisors Mark Clark

and Julie Zilles. Mark, for convincing me to come back to Illinois for my PhD and giving me the

independence required to start my ambitious project. Julie was an excellent advisor and helped

me navigate through my PhD with her steady advice and sympathetic ear. I know that I have

been very demanding of her time and attention and certainly, patience, many times; but she was

always there for me and supported me wholeheartedly. I did follow some crazy ideas and she let

me run through them logically and made sure I did not get distracted from the main goal of my

work. I cannot thank her enough and hope to be able to justify her faith in my abilities through

continued success.

Dr. Wolfgang Meier was a great collaborator, mentor, and committee member. I started

this project inspired by some of his work and he was very welcoming and supportive of me

learning techniques and working in his lab. The months I spent in his lab were probably the

most productive part of my PhD experience.

Dr. Snoeyink was a great committee member and mentor. He has helped me with great

professional advice over the years starting with when I first started graduate studies in 1998. He

was the reason for me to wait “at least 5 years in industry” before returning for a PhD. I think

this was geat advice and I think has served me well.

I would also like to thank my former group members David Ladner and Won-young Ahn

for contributing to my growth as a researcher and as a person. David taught me about careful

planning and independent thinking as well ass opening my eyes to what was possible for a

graduate student to do on his own. I admire his independent thinking and ability to get along

with people of all kinds while getting things done! Won-young helped me learn about the

promise of molecular modeling and how it could be applicable to my work.

My current group members, especially Michelle Marincel and Sean Poust have taught me

a lot about collaborating on a research group. I must have been a constant strain on them with

my incessant stream of advice on how to do things better and how to think independently. I hope

this was somehow helpful enough to them to counterbalance dealing with me on a daily basis.

Michelle has helped me think about how to design experiments and explain my ideas to make

them accessible. Sean has helped me learn how to think my ideas through with his independent

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ideas and ability and willingness to raise objections to my harebrained theories. Other members

of the Zilles research group have also taught me many lab techniques and helped me with ideas.

Dr. Meir’s research group has also helped me learn about block copolymers and their

uses. Mariusz Grzelakowski was my closest collaborator and has become a great friend. We can

hopefully collaborate on our slate of “unfinished ideas” in the future. Katarzyna Kita was a great

resource and friend and her frank and insightful comments helped me get work done efficiently

while I was in Basel. Fabian Itel visited our lab in summer 2008 and has been an ongoing

collaborator and friend since. His straight talk, and his ability to make me see his side of the

story while working on problems together has been a great learning experience. Dr. Andreas

Engel and his group in Basel got me started on 2D crystallization of membrane proteins and I am

thankful for their help and advice in this area over the last two years.

The EES faculty at Illinois, both current and past, are outstanding people in addition to

being great researchers and teachers. In particular I would like to mention Drs. Timm

Strathmann, Helen Nguyen, Eberhard Morgenroth, Kevin Finneran and Benito Marinas for their

help and advice over the course of my PhD.

I developed a great working relationships and friendships with EES students over the

years. In particular thanks to Marty Page for helping me out with UV experiments and for being

a great friend. The occupants (past and current) of 4125 deserve my gratitude for expanding my

knowledge and cultural training and for bearing with my habit of discussing ideas for longer than

necessary. These include Kim Milferstedt, Adrienne Menitti, Nick Weihardt, and Xinyu Zhang.

Derek Vardon worked in our lab, was an office mate, a neighbor and a great friend.

Don Cropek and Irene McAllister from CERL, Champaign helped me get the

experience of writing a successful proposal and have been great collaborators. Hopefully, we

can find a way of continuing our collaboration.

Shaoying Qi; our lab manager, has helped by being very supportive with equipment and

lab issues as well as being a great mentor and somebody who I have had great discussions about

what it means to be an engineer and a scientist.

Towards the end of my PhD I visited Dr. Tom Walz and his group and in working with

them I was able to figure some things out in weeks that I though would not be possible during

my PhD. I am thankful to them and hope that my postdoctoral work with them will be

productive.

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Steve Daley has been an excellent resource for budget issues and has really helped me

navigate these issues. His ability to find a solution to almost any budget situation is amazing.

Marvis Ozerk, the EES program coordinator helped with the many letters I had to obtain for

fellowships and such and also with other logistical issues. Sue Lowry was very helpful with

purchasing issues and Bobbi Vance helped with reimbursements.

The initial funding for this work was provided through a small but important “loan” from

the CEE department in the first year. A University Dissertation Travel grant provided the next

critical funding step for the summer following the first year for a very productive summer in Dr.

Meier’s group. Funding from the National Water Research Institute came at a critical time to

keep towards the end of this summer. Finally, fellowships from American Water Works

Association and Environmental Protection Agency towards the end of the second year allowed

support for stipends as well as travel. National Science Foundation funding starting at the

beginning of the third year for this project helped us equip our lab completely for making ptotein

polymer membranes and hiring a graduate student. US Arrmy CERL funded an additional

project on perchlorate removal that helped us expand the scope of the work that I had initiated

and helped hire another graduate student and a couple of undergraduate students.

Charlie Werth and my running/social group, the Wandering Elderbarries (especially

Shane, Peter, Barry, Joe, and Jack) were a source of much enjoyment, laughter, as well as sage

advice over the years. They made Saturday mornings a hoot and the trips to the relays we took

part in, memorable!

My parents, Virendra and Sushila Yadav, have instilled in me a love of learning and a

respect for teachers. Thank you for all that you have done for me. My dad was even more

excited than me at the prospect of me becoming a professor someday. Hopefully, I will be able

to justify your enthusiasm by being a good teacher. My brother, Chandra Prakash has always

encouraged me in my studies and I appreciate his advice over the years. My in-laws, especially

my mother-in-law, Annamma Mathew helped with the kids during the last year of my PhD. She

is an amazing woman and never seems to tire. I can only aspire to be as active as her at her age.

Anu, my dear wife, makes me complete; and was a major driving force for me striving to

do my best. She spent many months alone while I was traveling or just spending time in the lab.

Finally, she has given me the greatest gifts of my life, Arun and Sierra, our beautiful kids. I

thank her with all my heart.

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TABLE OF CONTENTS 1 INTRODUCTION AND OBJECTIVES................................................................... 1

1.1 WATER AND WASTEWATER TREATMENT MEMBRANES....................... 2

1.2 AQUAPORINS ................................................................................................... 12

1.3 BLOCK COPOLYMERS AS MIMICS OF BIOLOGICAL MEMBRANES.... 17

1.4 OBJECTIVES ..................................................................................................... 22

1.5 ORGANIZATION OF DISSERTATION........................................................... 23

2 HIGHLY PERMEABLE POLYMERIC MEMBRANES BASED ON THE

INCORPORATION OF THE FUNCTIONAL WATER CHANNEL PROTEIN

AQUAPORIN Z ........................................................................................................ 24

2.1 ABSTRACT ........................................................................................................ 24

2.2 INTRODUCTION............................................................................................... 25

2.3 RESULTS............................................................................................................ 27

2.4 DISCUSSION ..................................................................................................... 32

2.5 MATERIALS AND METHODS ........................................................................ 34

3 BIOLOGICAL ROLE FOR BACTERIAL AQUAPORIN GATING

DISCOVERED IN BLOCK COPOLYMER MEMBRANES .............................. 39

3.1 ABSTRACT ........................................................................................................ 39

3.2 INTRODUCTION............................................................................................... 39

3.3 RESULTS............................................................................................................ 42

3.4 DISCUSSION ..................................................................................................... 47


4 SELF-ASSEMBLY OF MEMBRANE PROTEINS IN BLOCK

POLYMERS.............................................................................................................. 53

4.1 ABSTRACT ........................................................................................................ 53

4.2 INTRODUCTION............................................................................................... 53

4.3 RESULTS............................................................................................................ 54

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4.4 DISCUSSION ..................................................................................................... 58


5 SUMMARY, IMPLICATIONS, AND FUTURE RESEARCH............................ 65

5.1 HIGH PERMEABILITY AQPZ RICH MEMBRANE CAN MAKE

IDEAL DESALINATION MEMBRANES AND HIGH SENSITIVITY

SENSORS. .................................................................................................................. 65

5.2 TRIBLOCK COPOLYMERS CAN BE USED AS A PLATFORM FOR

STUDYING MEMBRANE PROTEIN PROPERTIES.............................................. 69

5.3 BLOCK COPOLYMERS CAN BE USED FOR DETERMINING

PROTEIN STRUCTURE. .......................................................................................... 72

5.4 MEMBRANE VESICLES CAN BE USED FOR CONDUCTING

REACTIONS ON SURFACES, ENVIRONMENTAL REMEDIATION AND

CARBON CAPTURE................................................................................................. 76

5.5 CONCLUSION ................................................................................................... 80

6 REFERENCES.......................................................................................................... 81

APPENDIX A.................................................................................................................. 95

APPENDIX B .................................................................................................................. 98

APPENDIX C................................................................................................................ 101

AUTHOR’S BIOGRAPHY.......................................................................................... 109

1

1 INTRODUCTION AND OBJECTIVES

Synthetic membranes are important for environmental applications, particularly for water and

wastewater treatment. They have become more relevant as we are faced with a scarcity of fresh

water sources in many parts of the world. This water scarcity is further complicated by

contamination of existing freshwater supplies and the emergence of new, difficult-to-remove

pollutants (Koplin et al. 2002). Membrane desalination technologies, such as reverse osmosis

(RO), have come to the forefront as excellent technologies for developing new water sources

such as brackish surface and groundwater (Bohdziewicz et al. 1999), seawater (Matsura 2001),

and even recycled wastewater (del Pino and Durham 1999; Goldstein 2006; Judd 2006). RO is

also used for removal of several existing and emerging contaminants (Kimura et al. 2003;

Kimura et al. 2004). However, RO’s environmental footprint is coming under increasing

scrutiny, especially for seawater desalination (Einav et al. 2002). The pressure requirement for

RO is high, resulting in high energy consumption. This is a continuing hurdle to extensive

application of this technology. Additionally, some endocrine disrupting compounds (EDCs) and

pharmaceutically active compounds (PhACs) are not easily treated using RO (Kimura et al.

2004). With increasing concern regarding the impact of climate change on traditional water

supplies (Kundzewicz 2007), the development of alternate sources such as seawater and

reclaimed water is imperative. This has led to a focus on research into enabling technologies

such as RO in recent years.

There has been tremendous development in the efficacy and affordability of RO membranes in

the last three decades. However, limitations still exist on their selectivity, permeability and

energy efficiency. Cell membranes, on the other hand, can be considered ideal solute rejecting

membranes. Studying these membranes may help us improve existing RO membranes. They are

a composite filter consisting of exceptionally selective proteins known as membrane proteins that

transport water, specific ions (K+), or specific macromolecules. Cell membranes conduct

extremely efficient filtration while removing salts and other dissolved solutes. Furthermore, they

transport water at extremely high rates. Current high salt rejection RO membranes have a

permeability of around 3-4 x 10-12 ms-1Pa-1 (Ladner 2009), while membranes mimicking those

present in red blood cells or kidney tubules can reach ~ 4 x 10-11 ms-1Pa-1 (Walz et al. 1994).

This enhanced permeability is due to the presence of water channel proteins, Aquaporins, in

2

these membranes. Aquaporins have exceptional water transport properties, transporting as many

as three billion water molecules per second while excluding solutes (Agre 2004). They have

been found in species from all domains of life and are important for diverse physiological

functions involving water transport.

My work focused on developing a highly permeable membrane for desalination by

inserting Aquaporins into amphiphilic block copolymer membranes. These membranes are

proposed as alternatives to currently used synthetic polymeric RO membranes. As background

for this work, in this chapter I introduce water and wastewater membranes, RO membranes and

their structure, use and limitations during desalination, current efforts to improve RO

membranes, aquaporins, the relevance of important structural motifs and mechanism of transport,

amphiphilic block copolymers, and the concept of aquaporin-based block copolymer biomimetic

membranes.

1.1 WATER AND WASTEWATER TREATMENT MEMBRANES

Synthetic polymeric membranes are widely used for water and wastewater treatment in

environmental engineering. The most porous membranes have controlled pore sizes as large as

several microns. At the opposite end of the spectrum (nonporous membranes) there are no pores

per se, rather transport occurs between overlapping polymer strands. Separation by these

membranes is based primarily on solute size. Figure 1.1 illustrates the size range of membrane

separations, and the contaminants common in water and wastewater treatment.

As seen from Figure 1.1, these membranes are used for separation of dissolved and particulate

matter from water and wastewater. Low pressure membranes (microfiltration and ultrafiltration)

are used for separation of particulate matter while high pressure membranes (RO and

nanofiltration) are used for removal of dissolved materials such as ions and macromolecules.

Membrane treatment technologies pertinent to environmental engineering are summarized in

Table 1.1.

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Figure 1.1: Membrane processes used in water and wastewater treatment. (Note: All images used in this figure are from the public domain and do not require copyright for use. Cryptosporidium and Giardia image is from www.epa.gov/microbes/cpt_gda.htm ; Viruses: rotovirus top image from http://en.wikipedia.org/wiki/Rotavirus and adenovirus bottom image is from http://en.wikipedia.org/wiki/Adenoviridae ; Humic acid structure is from http://en.wikipedia.org/wiki/Humic_acid)

Of the technologies presented in Table 1.1, high pressure membrane technologies have

the highest power needs due to the mechanism of water transport and the necessity of

overcoming the osmotic pressure created by rejection of soluble material, in particular, salts. I

will refer to high pressure membranes (both Nanofiltration (NF) and Reverse Osmosis (RO))

collectively as RO in this document. Typical operating pressures for low pressure membranes

are either up to ~ 1 bar vacuum (-14.5 psi) or between 0.3 and 2.1 bar pressures (~5 to 30 psi),

while for high pressure membranes operating pressures can range from 7 to 21 bar (~100 -300

psi) (for nanofiltration and brackish water reverse osmosis) to 55-70 bar (~800-1000 psi) for

seawater desalination. Consequently, energy consumption for RO facilities are a large fraction

(up to 60%) of the operating costs (Kumar et al. 2005). The relevance of RO membranes is

growing rapidly in coastal areas as well in inland areas with marginal water quality. Hence the

http://www.epa.gov/microbes/cpt_gda.htm

http://en.wikipedia.org/wiki/Rotavirus

http://en.wikipedia.org/wiki/Humic_acid

4

pressure to improve the energy efficiency and thus the sustainability of the RO process has

increased in recent years. New ideas are needed to move this important technology further.

Table 1.1: Applications of membrane treatment technologies

Application Separation Source Membrane Type Technology

Water Treatment Particulate

and microbial

contaminant

removal

Groundwater,

surface water,

seawater

Microfiltration

(MF) and

Ultrafiltration

(UF)

Low Pressure

Membranes

Water Treatment

and Reuse

Dissolved

contaminant

and salt

removal

Tertiary treated

wastewater,

groundwater,

surface water

Nanofiltration

(NF) and Reverse

Osmosis (RO)

High Pressure

Membranes

Wastewater

Treatment

Biomass

separation in

bioreactor

Raw and

primary treated

wastewater

MF/UF Membrane Bioreactors

(MBR)

Desalination Salt and

dissolved ion

removal

Brackish

surface and

groundwater,

seawater

Cation/Anion

Exchange

Membranes

Electrodialysis

(ED)/Electrodialysis

Reversal (EDR)

The following subsections describe the structure of current RO membranes, the current

stage of development and their inherent permeability limitation. This is followed by inspiration

for a high permeability membrane derived from biological membrane protein channels.

1.1.1 Reverse Osmosis Membranes: Structure

Current RO membranes are either asymmetric or thin film composites (Lonsdale 1987;

Ho and Sirkar 1992; Cheryan 1998). Asymmetric RO membranes have a thin selective skin

layer supported on a porous sublayer of the same polymer. Asymmetric membranes are

synthesized using the phase inversion process in which a polymer solution in solvent is

immersed in a non-solvent bath. This leads to precipitation of the polymer into a polymer-rich

solid phase that forms the membrane and a polymer-poor liquid phase that forms the membrane

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pores or void spaces. Thin film composite (TFC) membranes, on the other hand, consist of a thin

polymer layer formed on one or more porous support layers. TFC membranes are made by

interfacial polymerization in which a porous polymer such as polysulfone is coated with a layer

of polymer or polymer components and then reacted using crosslinking agents. For both the

asymmetric and the TFC membranes, the surface layer determines the flux and separation. In

both cases the surface separation layer is a dense polymer layer about 100 nm thick. This has

implications on the energy efficiency for such membranes as described in the following sections.

1.1.2 Current state of development, limitations, and opportunities for improvement

There have been great improvements in the productivity and decrease in the cost of

synthetic membranes used for water and wastewater applications. Figure 1.2 presents a cost and

productivity index (cost per unit water production) for low pressure membranes and high

pressure membranes (specifically brackish water reverse osmosis (BWRO)) over the last few

decades. This index was calculated based on manufacturer-provided historical and projection

data. An explanation of this data is provided in Appendix A.

Figure 1.2: Cost reduction and productivity improvement in synthetic membranes used in water treatment. The indices present a cost/productivity value indexed to the first year data was available. Fits for UF/MF include two linear regions and for BWRO includes an exponential curve.

0

0.2

0.4

0.6

0.8

1

1.2

1989 1994 1999 2004Year

UF/MF Index

(a)

0

0.2

0.4

0.6

0.8

1

1.2

1980 1990 2000 2010

Year

BWRO Index

(b)

6

As discussed earlier, a critical need at present is to improve the efficiency of high

pressure membranes. Figure 1.2 (b) shows that the decrease in cost per unit productivity has

become relatively flat in recent years. Also researchers have pointed out that the permeability of

RO membranes has almost reached the thermodynamic limit of minimum energy required for

such separations (Zhu et al. 2009). Increases in productivity of such membranes are limited by

the mechanism of water transport through interfacially formed and crosslinked polymers. An

illustration of the mechanism of water transport in such polymer layers is provided in Figure 1.3.

The rate-controlling transport step is through the top interfacial thin film layer (shown in Figure

1.3 (a)). Figure 1.3 (b) shows the distribution of water within pores of the crosslinked structure

of this polymer. These pores are not connected in a static manner; hence RO membranes are

described as being nonporous. Water present in pores of such polymers diffuses through the

polymer layer when pores become interconnected by thermal motion of polymers (Kotelyanskii

et al. 1998; Kotelyanskii et al. 1999). This is called “jump” diffusion of water. Due to the

statistical nature of this phenomenon, it is inherently slow. This is particularly evident when the

scale of transport (~100 nm) is considered compared to the scale of each jump diffusion step (~

3Å) (Kotelyanskii et al. 1998). Also, water transport is related to the amount of crosslinking as

crosslinking reduces the possible connections between adjacent pores. Increasing amounts of

crosslinking also relates to higher salt rejection in polyamide RO membranes such as in high salt

rejection membranes (seawater RO membranes). This, in high rejection membranes the jump

diffusion rate is further decreased, leading to even lower transport rates.

Some natural (Aquaporin) and artificial (carbon nanotube) nanochannels transport water

more rapidly than existing RO membranes due to a fundamental difference in transport

mechanisms. Given the mechanistic constraints of RO water transport, it is instructive to look at

the mechanism of water transport in hydrophobic protein channels, in particular aquaporins and

their synthetic analogue, carbon nanotubes.

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Figure 1.3. Water transport through RO membranes is limited by the slow “jump diffusion” of water (a) Scanning Electron Micrograph of a Thin Film Composite (TFC) RO membrane. The top separating layer has delaminated from the support in this micrograph. (b) A molecular model of water distribution in a small part of the separating layer and pathway for “jump diffusion of water” as described in (Kotelyanskii et al. 1998) (c) Distribution of pores and water including the crosslinked polymer structure. Note: (b) and (c) are used with permission from (Kalinichev 2007).

Water transport in narrow hydrophobic channels (<100 nm) occurs by frictionless

movement of water molecules in a single file across the channel walls (Figure 1.3 (a) and (b))

across small lengths (10-20 nm). This frictionless single file movement of water molecules

down such channels, under the effects of an osmotic or pressure gradient (Majumder et al. 2005;

Hinds 2007), results in an enhanced transport rate that could be used for high permeability RO

membranes as described in later chapters.

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Figure 1.4: Water transport in narrow hydrophobic channels (a) and (b) are illustrations of water flow in Aquaporins and Carbon Nanotubes (CNTs) respectively. (c) and (d) are illustrations of the velocity profile in Poiseuille flow (parabolic) and in confined slip flow (blunted parabolic). An additional profile of classical Poiseuille flow is shown in (d) in light blue to indicate comparison to classical flow and show where zero velocity is in relation to the shown velocity profile.

The following discussion provides a simple illustrative description of water flow in smooth

hydrophobic channels such as aquaporins and CNTs. Here, a classical fluid dynamics

description is presented as a simple illustration of the major reason for flux enhancement seen in

such channels.

In confined narrow channels there is a deviation from the classical Poiseuille flow

leading to an apparent velocity, the slip velocity, at the channel wall. In order to account for slip

velocity the Poiseuille equation is modified as follows.

2 2

2

2( ) 1 ,

4sLR r P

u rR R z

where, u (r) is velocity of water at a radial distance r from the center,

R is the radius of the channel,

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Ls is the slip length at the solid liquid interface,

μ is the viscosity of water, and

P

z

is the pressure drop across the channel length z

The slip length is a characteristic scale that represents the discontinuity of the velocity

profile at the channel wall. In the above equation, the slip length is given by (described in

(Thomas et al. 2009))

( )

,s

r R

u rL

dudr

where, du/dr is the velocity gradient at r.

For CNTs, the slip length has been empirically determined to depend on carbon

nanochannel diameter and is given by (as described in (Thomas et al. 2009))

, 3( ) ,s s

CL d L

d

Where, Ls,∞ = 30 nm, the slip length over a flat grapheme (single layer molecular carbon

sheet) sheet, and C = 352 nm (a fitting parameter reported in (Holt et al. 2006)), and d is

diameter of the channel.

The effect of slip in these narrow hydrophobic channels is that the parabolic shape of the

velocity profile seen in Poiseuille flow is “blunted” (Figure 1.4 (b) and (c)) with decrease in

radius. This is illustrated using numerical calculations of the flow profile in Figure 1.5 (a).

Additionally as the channels become narrower and the slip length increases the velocity increases

dramatically when compared to average velocity in Poiseuille flow as shown in Figure 1.5 (b).

The preceding discussion applies to the case where the size of these channels are beyond

the continuum limit (>1.6 nm) for water flow. This size is much larger than AqpZ channel

“pores” (~0.23 nm at the narrowest part) and slightly larger than current CNT membranes (~1.8

nm) pores. However a simple continuum discussion of transport in smooth hydrophobic

channels provides insight into the fundamental mechanism of transport enhancement. Transport

enhancement becomes more pronounced at non-continuum scale in such membranes. However,

at the smaller non-continuum scale the description is somewhat complicated by the structure of

water in confined geometries as well as by coordinated motion of water molecules and can only

be described using molecular dynamics simulations.

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Figure 1.5. Comparison of velocity profiles in Poiseuille flow (R>200nm) to that in confined slip flow. (a) Velocity profiles normalized to the maximum velocity for different channel radii showing the change in profile from parabolic to blunt profile. (b) Velocity profiles across the channel where the y axis represents the ratio of the radial velocity, u(r), to the average Poiseuille velocity for different channel radii. Note the large increase in velocity (and hence flow) as the R value goes from >200 nm (2x avg. Poiseville velocity as expected in Poiseville flow) to 0.8 nm. (~580 x avg. Poiseville velocity). The above was calculated using the approach presented elsewhere (Thomas et al. 2009).

The efforts to develop high permeability RO membranes including membranes that utilize the

above approach are summarized in the following section.

1.1.3 Ongoing efforts for developing high permeability RO membranes

There are currently three main approaches being investigated for developing high

permeability RO membranes. These are: (1) thin film nanocomposite (TFN) membranes, (2)

carbon nanotube (CNT) membranes and (3) Aquaporin-based biomimetic membranes.

The thin film nanocomposite (TFN) membrane is a direct improvement on the current RO

membranes and improves water transport efficiency in the thin selective skin of TFC polyamide

RO membranes. This is achieved by adding zeolite nanoparticles during active layer formation.

Adding these nanoparticles is hypothesized to increase local order and structuring in these thin

selective layers (Jeong et al. 2007). In accordance with this hypothesis, TFN membranes have

increased smoothness, hydrophilicity and negative charge. These properties may explain the

approximately 100 to 200 % (up to two times) improvement in permeability (Jeong et al. 2007)

11

seen over conventional TFC RO membranes. Additionally, incorporating nanoparticles of

different compositions and at different amounts provides control over mechanical, transport and

surface properties of such membranes (Hoek et al. 2008; Lind et al. 2009). The membranes are

currently being commercialized for implementation as full scale membrane modules.

Performance data on these modules are currently unavailable.

CNT membranes rely on the use of aligned, single wall carbon nanotubes in an impermeable

matrix. Currently synthesized CNTs have diameters in the range of ~ 1.8 nm or higher. There

are two commonly used approaches to synthesizing aligned carbon nanotubes that may be

suitable for membrane applications (Noy et al. 2007). Holt et al have developed a low pressure

chemical vapor deposition process to encapsulate a vertically aligned array of CNTs in silicon

nitride (Holt et al. 2006). After encapsulation the ends of the CNTs are opened up using an

etching process. Another approach to synthesizing aligned CNTs (Kim et al. 2007) uses a

polymer matrix. CNTs are first functionalized with amine groups and then dispersed in the

solvent - tetrahydrofuran (THF). This suspension is filtered through a hydrophobic Poly Tetra

Fluoro Ethylene (PTFE) filter to align the CNTs. A polymer solution of polysulfone is spin

coated to make the support matrix. Molecular dynamics and initial reports have shown great

promise for desalination using narrow CNT membranes (<0.5 nm). However, currently

synthesized CNT membranes have the major drawback of not being able to remove small ions

such as sodium and chloride. This is due to the fundamental difficulty of making carbon

nanotubes smaller than the hydrated diameter of these ions (Na+ has a diameter of ~0.2 nm).

Attempts to make narrower CNTs and functionalization of the internal surface of these CNTs are

being considered as options to improve ion rejection. However, no current publications report

success in making salt rejecting CNTs.

While the work presented in this thesis resulted in the first publication on AQP-based

membranes, to my knowledge three other research groups are actively pursuing similar ideas.

Most of the other ideas regarding the use of Aquaporin-based membranes prior to 2010 are

presented in patents. A research group at the Technical University of Denmark (as a part of a

European Union project called Membaq and a company Aquaporin AS) has patented an AQP-

based water treatment approach that utilizes lipid membranes (Jensen and Keller 2006; Jensen

2007). The Aquaporin AS group has recently published work on the development of a

microporous support for painting lipid bilayers or PMOXA-PDMS-PMOXA based monolayers

12

where aquaporins insertion was demonstrated (Gonzalez-Perez et al. 2009; Vogel et al. 2009).

This support is manufactured by laser ablation of an Ethylene Tetra Fluoro Ethylene (ETFE)

polymer film. They have shown insertion of several membrane proteins. However functional

insertion of Aquaporins has not been demonstrated at present. Montemagno et al have proposed

using triblock copolymers for protein insertion and using vesicles embedded in a polymer matrix

for water treatment (Motemagno 2006). Montemagno et al have not published this work,

although the group has patented several schemes for use of membrane proteins embedded in

block copolymer membranes (Montemagno et al. 2007). Kaufmann et al have recently published

a method to reliably coat a NF membrane with a lipid bilayer by depositing a tuned mixture of

lipids on the surface (Kaufman et al. 2010). They have demonstrated that the lipid bilayer is

continuous and could be a platform for insertion of Aquaporins. However, insertion of

Aquaporins and permeability of such membranes has still not been demonstrated. The rest of

this dissertation will discuss my results and ongoing efforts to develop Aquaporin rich

membranes for developing desalination membranes, as well as related findings on fundamental

properties and other possible applications of membrane proteins in block copolymers. Currently,

work on AQP-membranes in the other groups is focused on achieving protein insertion and on

development of larger scale membranes in order to directly measure permeability. Additionally,

work is ongoing on developing an appropriate support for such membranes. AQP-based

membranes have exceptional promise for both high permeability and high selectivity. However

this technology is at an early stage.

1.2 AQUAPORINS

Aquaporins are a class of membrane-spanning proteins that primarily transport water

across the cell membrane (Agre 2004). Their existence had been hypothesized for many decades

due to observation of anomalously large fluxes across some biological membranes such as

erythrocyte plasma membranes (summarized in (Finkelstein 1987)). These fluxes could not be

solely explained by diffusion across the cell membrane, so the presence of water channel

proteins was proposed. Agre and coworkers first demonstrated the water channel activity of a 28

KDa erythrocyte membrane protein expressed in Xenopus laevis oocytes (Preston et al. 1992).

This was the human aquaporin subsequently named AQP1. Since then 10 other

human/mammalian aquaporins have been discovered (named AQP0 through AQP10) with

13

critical physiological functions (Agre and Kozono 2003). Additionally, aquaporins have been

found in plants, yeast, bacteria and archaea (Calamita et al. 1995; Maurel 1997; Bonhivers et al.

1998; Kozono et al. 2003). Most aquaporins have functions related to water transport but solute

transport (reviewed in (King et al. 2004)) as well as formation of intercellular junctions (AQP0,

AQP4) by aquaporins have also been reported (Engel et al. 2008).

Aquaporins have a unique structure that allows them to transport water while excluding all

other solutes including protons. This flow is in the direction of the osmotic gradient. They have

an hourglass architecture and “pore openings” of around 3 Å lined with mostly hydrophobic

amino acids. They have excellent solute rejection properties. The mechanisms responsible for

solute rejection are summarized in Figure 1.6 (de Groot and Grubmuller 2001; Kozono et al.

2002). Aquaporins have 6 membrane spanning domains that are composed of alpha helices

(Figure 1.7) connected by 5 loops (A through E). Two of these domains (loop B and E) dip

halfway into the center of the hydrophobic core of the lipids, forming a hour glass structure that

provides a path for water (Jung et al. 1994). Both these loops contain a universally conserved

asparagine-proline-alanine (NPA) motif where the loops fold into the center of the membrane.

These two sets of NPA motifs form a part of the selectivity mechanism of the Aquaporin

depicted in Figure 1.6. The presence of a charged arginine residue close to this region and

interacting with water molecules passing down the lumen (pore) of the aquaporin in a single file

is responsible for inhibiting proton transport across aquaporins. There are several explanations

for proton diffusion in water. For example the Grotthuss mechanism (Agmon 1995) suggests

transfer via hydrogen bonds and transient hydronium ions. Thus a single file of water molecules

would be expected to efficiently transport protons by acting as a proton wire. This is indeed the

case in channels such as Gramicidin A where such an unbroken proton wire is invoked (Pomes

and Roux 2002). However, due to the presence of the two sets of NPA residues and a conserved

Arginine residue (Arg 189 in AqpZ, Arg 185 in AQP1) in a defined configuration, every water

molecule passing through this region has to form a series of hydrogen bonds with the aquaporin

residues in a manner that leads to reorientation of the water molecule, thus breaking the proton

wire (Murata et al. 2000). As first seen from molecular dynamics simulations and eventually

experimentally confirmed by X-ray crystallography, the water molecules face different directions

in the two halves of the protein (de Groot and Grubmuller 2001; Tajkhorshid et al. 2002).

14

Aquaporin exists as a tetramer in its native state; however each monomer is functional on its

own.

Figure 1.6: Mechanisms for rejection of solutes and transport of water through aquaporins illustrated using AQP1 as a model. The amino acid residues important for these rejection mechanisms are highlighted. Histidine 180 (His 180) and Arginine 185 (Arg 185) form the narrowest part of the channel (2.8 Å) and this size restriction responsible for size exclusion of larger solutes. Arg 185 is positively charged and the presence of this residue at the narrowest part of the channel helps reject positively charged solutes by electrostatic repulsion. Water molecules undergo a molecular reorientation by hydrogen bonding with a residue near the pore (Murata et al. 2000; de Groot and Grubmuller 2001; Chakrabarti et al. 2004). This molecular reorientation (water dipole reorientation) is responsible for preventing passage of H+ ions by breaking extensive hydrogen bonding that normally allows rapid H+ transfer in bulk water. The above illustration is based on Kozono at al (Kozono et al. 2002) and Jung et al (Jung et al. 1994) and was recreated using an example simulation (trajectory file from a molecular dynamics simulation of water passage through AQP1 (Grabmueller 2010)).

15

Figure 1.7: Predicted topology and sequence of AqpZ (a) based on (Borgnia et al. 1999). The transmembrane domains are shown as tubes and are numbered 1 through 6 starting from the N-terminus. The loops connecting these domains are shown as ribbons and numbered A through E. The highly conserved NPA residues are shown on loops B and E. It can be seen that this structure is symmetrical around loop C. (b) shows the folding of this polypeptide chain into a channel with the intracellular loops orienting parallel to each other in one of two tilt angles. (c) and (d) are side and top views of AqpZ monomer shown in ribbon representation.

The bacterial Aquaporin, Aquaporin Z (AqpZ) was selected for this study because of the

ease of laboratory expression and purification of this protein in its native host, Escherichia coli.

Additionally, this protein has been reported to be robust under various solution conditions and

active upon reconstitution into lipid vesicles (Borgnia et al. 1999). It also has one of the highest

water permeabilities of aquaporins that have been tested (Hashido et al. 2005). The sequence

16

and predicted topology (structure and layout in the membrane) (based on (Borgnia et al. 1999))

as well as the determined structure of AqpZ are shown in Figure 1.7.

As can been seen from the placement of the six transmembrane domains of Aquaporin

and its location within the membrane, the lipid layer is required to stabilize the structure of the

protein. The hydrophobic interactions between the transmembrane parts of the protein and the

hydrophobic core of the lipid bilayer are important for correct orientation and stability of

aquaporins. This is illustrated in Figure 1.8. All membrane proteins have hydrophobic

transmembrane regions, usually in the form of a band on its outer surface. In the absence of a

hydrophobic “cushion” around the protein, interactions between proteins lead to their

precipitation and inactivation. These cushions most commonly are made from lipids. During

purification of membrane proteins these lipid cushions are replaced by appropriate amphiphilic

detergent molecules. In order to utilize the function of these proteins they have to be placed in a

lipid bilayer-like environment. Such a lipid-like environment is provided in this work by

utilizing block copolymers that self assemble into structures that allow for membrane protein

insertion.

Figure 1.8: Schematic of the lipid bilayer with inserted channel protein (transmembrane protein) (a) shows the insertion of the channel into the lipid bilayer with the pink region representing the hydrophobic surface of the protein that interacts with the hydrophobic core of the membrane bilayer formed from the tails of the lipid molecules illustrated in (b). (c) depicts the hydrophobic residues on the AqpZ monomer (in surface representation) in pink as an illustration of the hydrophobic band in membrane proteins.

17

1.3 BLOCK COPOLYMERS AS MIMICS OF BIOLOGICAL MEMBRANES

Amphiphilic block copolymers can self assemble into structures mimicking lipid bilayers in

aqueous solutions. Such block copolymers are synthesized as diblock co-polymers with one

hydrophilic block and one hydrophobic block linked together by a covalent bond or triblock co-

polymers with two hydrophilic end blocks and a hydrophobic middle block. These block

copolymers assemble into a variety of structures in aqueous solutions, including micelles, rods

and vesicles depending on the amount of water present (Discher and Eisenberg 2002). The

morphology seen in such systems is a result of hydrophobic interactions between the polymer

molecules and the shape of individual polymer molecules; this is dictated by the ratio of the

hydrophilic to hydrophobic block as well as their individual conformations (Zhang and

Eisenberg 1995).

1.3.1 Amphiphilic block copolymers

Block copolymers can be designed to have hydrophobic and hydrophilic blocks. In a solvent

selective for one of these blocks, polar solvents for the hydrophilic block and nonpolar solvent

for the hydrophobic block, one of the blocks interacts favorably with the solvent while the other

block is shielded leading to a phase separation at the scale of the hydrophobic or hydrophilic

blocks. This phase separation occurs while being constrained by the covalent bond between the

blocks and in some cases leads to formation of monolayers or bilayers that mimic the structure of

the lipid bilayer. These amphiphilic molecules aggregate into structures such as vesicles and

form an alternative platform for insertion of membrane proteins. These protein-polymer

aggregates can then be utilized for studying membrane protein properties and for making new

engineering materials. The following paragraphs and subsections provide a brief introduction to

block copolymer properties and summarize available literature on insertion of membrane


Amphiphilic block copolymers were used in this study as biomimetic membranes. They

provide several advantages over artificial and synthetic lipids in engineering applications. These

include:

i) High mechanical and chemical stability when compared to lipid membranes. Lipids have

the following disadvantages when used for applications (reviewed in (James C-M. Lee

2001)). Lipid membranes cannot be stretched beyond 5% of their original area without

18

rupture under osmotic or other stresses (Needham and Zhelev 1996). Because of

susceptibility to thermal phase transitions the permeability of lipid membranes is highly

variable, leading to solute leakage (Needham and Zhelev 1996). Block copolymer

membranes, on the other hand, perform much better than lipid membranes in terms of rupture

strength (toughness) (Discher et al. 1999) and thermal stability (James C-M. Lee 2001). The

cohesive energy density or toughness for polyethylethylene-polyethyleneoxide (EE-EO)

block copolymer membranes are 5-50 times higher than lipid membranes (Discher et al.

1999). Liposomes can also be strongly affected by pH, ionic strength, and temperature (Grit

and Crommelin 1993), complicating studies that need to be conducted under these conditions.

In this work, I show that the PMOXA-PDMS-PMOXA block copolymer vesicles are

insensitive to extreme pH values (between 2 and 12) and high ionic strengths (of up to 1 M).

They could also be engineered to be sensitive (Sauer et al. 2001) to pH changes.

ii) Low permeability for water (James C-M. Lee 2001; Kumar et al. 2007) and gases (ongoing

work, see Summary and Conclusions section) while being impermeable to charged species.

The structure and function of membrane transport proteins, including aquaporins, are typically

studied via reconstitution in a lipid matrix. However, lipids have high permeability for water

and gases, making it difficult to study transport of these molecules (Verkman 2002; Hill et al.

2004). The low permeability of block copolymers enhances the difference in transport

behavior between the membranes with and without transport proteins and allows sensitive

measurement of transport rates of the inserted transport proteins.

iii) Customizable properties. They can be engineered to provide the desired properties such as

fluidity or morphology by the choice of blocks and block lengths (Discher and Eisenberg

2002) or their length ratios (Zhang and Eisenberg 1995). Additionally, the choice of blocks

can provide a range of properties to these membranes. A highly relevant example is the use of

PMOXA as the hydrophilic block which provides the possibility of adding antifouling

properties to proposed desalination membranes and other applications (Konradi et al. 2008)

where protein deposition is undesirable.

iii) Ability to perform end group functionalization. Their end groups can be modified (Napoli

2008) by molecules such as biotin (for recognition and immobilization) (Grzelakowski et al.

2009), methacrylate (for stabilization by crosslinking) (Nardin et al. 2000), fluorescent

19

molecules (for imaging) (Grzelakowski et al. 2009) and even drugs (for drug delivery) (Peer

et al. 2007).

The amphiphlic block copolymers used in this work were poly-(2-methyloxazoline)-

poly-(dimethylsiloxane)- poly-(2-methyloxazoline) (PMOXA-PDMS-PMOXA) based

triblock polymers (Nardin et al. 2000). The general structure of this polymer is shown in

Figure 1.9.

Figure 1.9: Structure of the PMOXA-PDMS-PMOXA polymers used in this study

1.3.2 Membrane protein insertion into block copolymer membranes

Self assembled block copolymers in aqueous solutions, in particular the PMOXA-PDMS-

PMOXA system, have been studied over the last decade with a focus on insertion of membrane

proteins and realization of biochemical reactions within vesicle. Table 1.2 presents a summary

of the studies conducted with block copolymer systems and membrane proteins.

All the reported studies shown in Table 1.2 have used triblock copolymers with the same middle

block – Polydimethylsiloxane (PDMS). All the studies discussed below are referenced in the

above table. The most common hydrophilic block was polymethyl oxazoline (PMOXA).

Polyethyl oxazoline (PEtOz) was used in one study while another study used Polyethylene oxide

(PEO) as one of the hydrophilic blocks. The inserted proteins include the bacterial outer

membrane proteins OmpF, LamB, and FhuA, the bacterial water channel protein AqpZ, the

bovine eye lens aquaporin (AQP0), the purple membrane H+ pump bacteriorhodopsin (bR), the

cytochrome oxidase (COX), the bacterial Fo-F1 ATPase and the NADH-ubiquinone

Oxidoreductase. Of the above, all proteins (except AQP0) were shown to be active in this block

copolymer.

N NSi

O OSi Si

n OOHHO

O m m

Polydimethylsiloxane Poly‐2‐methyl‐2‐oxazoline Poly‐2‐methyl‐2‐oxazoline

Hydrophilic

Hydrophobic

20

Table 1.2: Summary of literature on membrane protein insertion in block polymers

These examples show that the PDMS middle block is appropriate for insertion of

membrane proteins. This is in spite of the large mismatch between the size of this hydrophobic

block and the length of the hydrophobic band on most membrane proteins. In lipid bilayers the

hydrophobic region is approximately 4 – 5 nm, which matches the hydrophobic regions on the

Block Polymer system Type Study focus Membrane

protein

Reference

Polymethyl oxazoline

(PMOXA) -Polydimethyl

siloxane (PDMS)-PMOXA

ABA Vesicle formation,

crosslinking

None (Nardin et al. 2000)

PMOXA-PDMS-PMOXA ABA Antibiotic

transport

OmpF (Nardin et al. 2000)

PMOXA-PDMS-PMOXA ABA Virus assisted

DNA loading into

vesicles

LamB (Graff et al. 2002)

PMOXA-PDMS-

Polyethylene oxide (PEO)

ABC Directed insertion

of membrane

protein

AQP-0 (Stoenescu et al. 2004)

PMOXA-PDMS-PMOXA ABA Proton transport bR and COX (Ho et al. 2004)

Poly Ethyl Oxazoline

(PEtOz)-PDMS-PEtOz

ABA Synthetic ATP

production

bR and Fo-F1

ATPase

(Choi and

Montemagno 2005)

PMOXA-PDMS-PMOXA ABA Aquaporin activity

measurement

AqpZ (Kumar et al. 2007) –

this work

PMOXA-PDMS-PMOXA ABA Reduction

triggered channels

FhuA (Onaca et al. 2008)

PMOXA-PDMS-PMOXA ABA Immobilized

nanoreactors

OmpF, enzyme

encapsulation

(Grzelakowski et al.

2009), also see chapter

5 for my contributions

PMOXA-PDMS-PMOXA ABA hydrophobic block

length effect on

protein activity

NADH-

ubiquinone

Oxidoreductase

(Graff et al. 2009)

PMOXA-PDMS-PMOXA ABA Activity of

encapsulated

enzymes

Encapsulated

superoxide

dismutase

(Onaca et al. 2010)

21

outside of most membrane proteins (Figure 1.8). A large hydrophobic mismatch between these

two regions might lead to an energetic penalty and limit the stability of such self assembled

systems. At first glance, such seems to be the case in the block copolymer membranes, where

the hydrophobic region is around 10 nm. However, Pata and Dan (Pata and Dan 2003) have

suggested that the flexibility of the hydrophobic block as well as its polydispersivity could lead

to regions immediately adjacent to the membrane proteins matching the hydrophobic band of the

membrane protein. This is illustrated in Figure 1.10(a). PDMS is a highly flexible molecule

and may be subject to such a phenomenon. Figure 1.10(b) presents a conceptual model of how

this principle would result in a block copolymer membrane with inserted aquaporins. Regardless

of the mechanism of stable aquaporin insertion in these membranes, the measured activity

indicates that we have obtained functional incorporation.

Figure 1.10: Possible mechanism of insertion of membrane proteins in thick block copolymers. (a) and (b) Flexible middle blocks are usually highly stretched and could shrink up to half their length to accommodate membrane proteins with sizes that are half the length of the hydrophobic block without excessive energetic penalties (Pata and Dan 2003). (c) A conceptual model for a PMOXA-PDMS-PMOXA membrane with inserted membrane proteins based on the above

22

mechanism, from (Mecke et al. 2006) (http://dx.doi.org/10.1039/b605165k), Reproduced by permission of The Royal Society of Chemistry.

As demonstrated in my work, the functional insertion of AqpZ in block copolymers, the

stability of block copolymers, and the high permeabilities and selectivity expected suggest AqpZ

molecules inserted into PMOXA-PDMS-PMOXA block copolymers may be an excellent starting

point for development of high efficiency biomimetic desalination membranes.

1.4 OBJECTIVES

The main hypothesis of this work is that efficient desalination membrane can be developed

by insertion of the bacterial aquaporin, AqpZ into PMOXA-PDMS-PMOXA block copolymers.

The specific objectives and a brief summary of the work conducted under each objective are

provided below.

o Permeability measurements of ABA type PMOXA-PDMS-PMOXA block copolymer

vesicles with and without AqpZ to demonstrate enhanced permeability of membranes with

incorporated AqpZ (Chapter 2). This chapter discusses proof of principle for using AqpZ

inserted in block copolymers as desalination membranes. Here I show that up to 3000

times increase in permeability can be obtained by inserting Aquaporins in block

copolymers while maintaining selectivity. The permeabilities measured for these AqpZ-

ABA membranes were an order of magnitude or more above that current commercial

membranes.

o Determination of pH behavior of such AqpZ-ABA membranes (Chapter 3). In this chapter

the discovery of low pH-induced gating of bacterial aquaporin is reported. In particular,

the use of block copolymer enabled the measurement of permeability of AqpZ over a

wide pH range. This is in contrast to lipid membranes commonly used for such studies,

which are unstable at low pH values. This work demonstrates the presence of aquaporin

gating in bacteria and discusses its physiological significance.

o Development of a flat Aquaporin rich membrane by using an interfacial templating

monolayer (Chapter 4). In this chapter a templating polymer monolayer was used to

arrange histidine-tagged aquaporins. This was further used to assemble AqpZ-rich

triblock copolymer films under this template. I was successful in making small, protein-

http://dx.doi.org/10.1039/b605165k

23

packed films that are crystalline and may have relevance to protein structure

determination in block copolymers. I also observed what may be 2D crystallization of

the PMOXA-PDMS-PMOXA polymer alone or in a unique manner (distinct from

crystallization in lipids) with AqpZ. This has not been reported before for any polymer

using the detergent removal method employed here. These crystals need to be studied

further but may be microporous materials with unique properties.

1.5 ORGANIZATION OF DISSERTATION

This dissertation is organized into an introduction, followed by papers on three specific areas of

membrane proteins in block copolymers (Chapters 2 through 4 introduced above). The final

section summarizes my dissertation work with focus on a) broader implications of the conducted

work, b) related projects that were inspired by and initiated during the course of this work and to

which I contributed, and c) proposed future work that could be conducted to further develop the

ideas developed in this dissertation and associated projects. The appendix contains additional

information and details on an approach to characterize crosslinking in methacrylated PMOXA-

PDMS-PMOXA polymers to determine their stability.

24

2 HIGHLY PERMEABLE POLYMERIC MEMBRANES BASED ON THE

INCORPORATION OF THE FUNCTIONAL WATER CHANNEL PROTEIN

AQUAPORIN Z

Ackowledgements: I performed all the experiments described in this chapter except for

PMOXA-PDMS-PMOXA polymer synthesis, transformation of the ptrcHis10AqpZ plasmid into

E.coli and cryo-electron microscopy.

The results described in this chapter have been published. The citation is as follows: Kumar, M;

Grzelakowski, M; Zilles, J; Clark, M; Meier, W. 2007. Highly permeable polymeric membranes

based on the incorporation of the functional water channel protein Aquaporin Z, Proceedings of

the National Academy of Sciences. 2007;104(52):20719 – 20724. All figures, tables and text are

reproduced from this published work. Copyright (2007) National Academy of Sciences, U.S.A.

Explicit permission is not required as I am author of this work.

2.1 ABSTRACT

The effects of incorporation of the bacterial water channel protein, Aquaporin Z (AqpZ) on

permeability and solute transport characteristics of highly impermeable amphiphilic triblock

polymer vesicles were investigated. These vesicles were made of a novel polymer with

symmetric poly-(2-methyloxazoline)-poly (dimethylsiloxane)-poly (2-methyloxazoline)

(PMOXA15-PDMS110-PMOXA15) repeat units. Permeability measurements were conducted by

measuring light scattering of the polymer vesicles placed under an outwardly directed osmotic

gradient of salt in a stopped flow apparatus. These polymer vesicles were found to be highly

impermeable. However, a large enhancement in water productivity (permeability per unit

driving force) of up to ~800 times that of pure polymer was observed when AqpZ was

incorporated. The activation energy (Ea) of water transport for the protein-polymer vesicles (3.4

kcal/mol) corresponded to that reported for water-channel-mediated water transport. The solute

reflection coefficients of glucose, glycerol, salt and urea were also calculated. The productivity

values obtained for AqpZ-incorporated polymers were an order of magnitude or larger than for

existing salt-rejecting polymeric membranes. This approach represents a exciting new direction

for developing more efficient water treatment membranes for municipal and medical

25

applications. The distinct levels of permeability obtained by using different concentrations of

AqpZ may provide a key property for drug delivery applications.

2.2 INTRODUCTION

Biological membranes have excellent water transport characteristics, with certain membranes

able to control permeability over a wide range. Membranes such as those present in the proximal

tubules of the human kidney (Knepper et al. 1996) be induced to insert specific water channel

membrane proteins known as Aquaporins (AQPs) to increase permeability. Other biological

membranes, such as those in mammalian optic lenses (Gorin et al. 1984), erythrocytes (Preston

et al. 1992), and many other cell membranes(Nielsen et al. 1993), are constitutively AQP-rich.

The permeabilities observed in AQP-rich membranes are orders of magnitude higher than that

observed for unmodified phospholipid membranes (Borgnia et al. 1999). Additionally, some

members of the AQP family have excellent solute retention capabilities for very small solutes

such as urea, glycerol and glucose even at high water transport rates (Meinild et al. 1998;

Borgnia et al. 1999). These properties result from the unique structure of the water-selective

AQPs. AQPs have six membrane-spanning domains and a unique hourglass structure (Jung et

al. 1994) with conserved charged residues that form a pore that allows the selective transport of

water while rejecting solutes. The AQP used in this study was a bacterial Aquaporin from

Escherichia coli – Aquaporin Z (AqpZ). AqpZ was selected as it has been shown to enhance

the permeability of lipid vesicles by an order of magnitude (Borgnia et al. 1999)while retaining

small uncharged solutes(Borgnia et al. 1999). Additionally, AqpZ can be expressed in relatively

large quantities in E. coli and has been reported to be quite stable under different reducing

conditions (Borgnia et al. 1999) and at temperatures of 4 ° C for extended periods of time-

properties that make it attractive for applications in drug delivery and water treatment.

The AQP’s high permeability and high specificity could be very valuable for a variety of

applications. High permeabilities and excellent solute retention of small solutes are important

for water treatment in critical medical applications such as dialysis (Pastan and Bailey 1998;

Pontoriero et al. 2003) as it could lead to reduced equipment size and more efficient use of

energy. A significant improvement in the permeability of solute rejecting membranes would be

a large step in improving the economics of desalination for municipal applications as well.

Desalination is becoming increasingly important for water production in semi-arid coastal

26

regions as well as for wastewater recycling (Service 2006; Tal 2006). Reverse Osmosis (RO)

membranes are most commonly used for this application and the use of Forward Osmosis (FO)

membranes is creating substantial interest (Patel-Predd 2006; Service 2006). However, the use

of RO requires large consumption of energy while FO applications need large membrane areas.

This is due to the low productivity of currently used commercial RO and FO membranes.

Finally, the ability to produce membranes that can be designed to have low initial permeabilities

is desirable for drug delivery applications (Discher et al. 1999); however, a high level of control

over this permeability could be an additional advantage (Dinsmore et al. 2002).

The effects of AQPs on the permeability of biological and synthetic lipid membranes has been

studied by incorporating these proteins into liposomes (Borgnia et al. 1999), frog oocytes

(Preston et al. 1992), and cellular secretory vesicles (Coury et al. 1996). However, the direct use

of biological membranes or synthesized lipid membranes for water treatment and drug delivery

applications has practical disadvantages. The major limitation is the low stability of lipid

membranes (Duncan 2003). Obtaining and processing large volumes of such membranes would

also present technical challenges.

The use of synthetic polymers, in particular lipid-bilayer-like amphiphilic block copolymers, is

exciting in this regard, as they could provide the necessary chemical and mechanical stability

while still providing a amphiphilic structure that allows incorporation and function of membrane

proteins such as AQPs (Taubert et al. 2004). Some membrane proteins have been functionally

inserted into block copolymer membranes (Meier et al. 2000; Graff et al. 2002; Choi and

Montemagno 2005; Choi et al. 2006). These include the beta barrel OmpF protein, the bacterial

receptor protein LamB and bacteriorhodopsin. Directed insertion of the eye lens AQP-

Aquaporin 0 (Aqp-0) into similar block copolymer membranes has been also been demonstrated

(Stoenescu et al. 2004), although this initial study did not examine function. Additionally,

studies on the permeability of block copolymer membranes are few dd and no previous studies

have reported on the magnitude of improvement in permeability that could be obtained in block

copolymer membranes by insertion of AQPs. Data on rejection of dissolved ionic solutes such

as salt (NaCl) has also not been reported before for AQP-rich membranes.

To investigate the potential of protein polymer membranes for water treatment applications,

solute and water transport properties as well as physical characteristics were analyzed for a

specific protein-polymer membrane. This membrane was composed of a novel triblock

27

copolymer with inserted AqpZ and is referred to throughout the text as an AqpZ-ABA

membrane. The composition of the symmetric triblock copolymer was PMOXA15-PDMS110-

PMOXA15. This new ABA polymer has a large hydrophobic block (110 PDMS groups) that is

expected to result in more mechanical stability and low permeability. The physical

characteristics of the ABA triblock copolymer were studied using microscopy techniques, while

light scattering was used to characterize the permeability.

2.3 RESULTS

2.3.1 Synthesis of AqpZ-ABA polymer membranes

Histidine-tagged AqpZ was overexpressed in Escherichia coli and purified using nickel affinity

chromatography (Sulkowski 1985; Borgnia et al. 1999). A large yield of pure protein (between

2.5 and 15 mg/L of culture) was obtained for three different purification runs, indicating the

potential for large yields using the procedures described. PMOXAm-PDMSn-PMOXAm (ABA)

triblock copolymers were synthesized using a ring opening cationic polymerization procedure.

Polymer vesicles were produced using the film rehydration method described in detail in the

methods section.

2.3.2 Characterization of Polymer Vesicles

To determine the permeability and solute rejection properties of polymer vesicles using stopped

flow spectroscopy a knowledge of the physical dimensions (radius in hydrated state) and

morphology (hollow versus solid sphere structure) is necessary. ABA polymer vesicles were

therefore characterized using static and dynamic light scattering, Transmission Electron

Microscopy (TEM), cryo-TEM, and Atomic Force Microscopy (AFM).

Using dynamic light scattering, a hydrodynamic radius (Rh) of approximately 160 nm was

estimated. This size is consistent with similar PMOXA-PDMS based block copolymers with

shorter hydrophobic blocks (Nardin et al. 2000). The ratio of the radius of gyration (Rg) from

static light scattering experiments to the hydrodynamic radius from dynamic light scattering

was ~1 (Rg/Rh = 1.03) and supports a hollow sphere morphology (Nardin et al. 2000). The

molecular weight of each vesicle was estimated at 52x106 g/mol using static light scattering.

28

Transmission Electron Microscopy (TEM) and cryo TEM were also conducted on these

particles. The TEM images show relatively uniform sizes for the different vesicles imaged.

From cryo TEM, the radius of the vesicles in the hydrated state was 117 nm.

Figure 2.1 shows representative images obtained from electron microscopy. Radii estimated

from cryo TEM were used in further calculations of permeability as this method best preserves

the structural features of vesicular structures (Almgren et al. 2000).

The presence of spherical vesicle like aggregates with a hydrophilic corona was further

supported by AFM measurements. During AFM, when the suspension of dilute vesicles is

spread on a hydrophilic mica surface, a film, punctuated with emerging vesicles, was seen to

form (Figure 2.1).

Figure 2.1: Examination of polymer vesicles using microscopy. The cryo TEM images were used for size determination as regular TEM and AFM influence the structure of the observed vesicles. (a) cryo transmission electron micrograph of a ABA polymer vesicle.(scale bar 200nm) (b) Electron micrograph of a cluster of vesicles (scale bar 50 nm) (c)Atomic force micrograph of vesicles on mica in non tapping mode shows that a film of polymer is formed on the hydrophilic mica surface with vesicles located in the films.

2.3.3 Permeability Measurements

The permeabilities of ABA and AqpZ-ABA vesicles were investigated using stopped flow light

scattering experiments as described in Borgnia et al, 1999. Vesicle suspensions were rapidly

mixed with osmotic solutions (1.7 osmol/L) of salt (NaCl). The shrinkage of these vesicles was

followed by monitoring the increase in light scattering with time. The initial rise in the

experimental data was fitted to an exponential rise equation, and the exponential coefficient (k)

was used in calculating permeability.

29

These results are shown in Figure 2.2 for the ABA and AqpZ-ABA vesicles with a protein to

polymer molar ratio of 1:200. For AqpZ-ABA vesicles the time scale for the exponential rise

was between 5 and 20 milliseconds, while up to 10 seconds were required to capture the

exponential rise in light scattering of ABA vesicles because of its low permeability. The

calculated permeabilities of the ABA and the AqpZ-ABA vesicles were 0.8 m/s and 74 m/s

respectively based on these results. This represents a large permeability increase of ~ 90 times

with protein incorporation. Assuming complete incorporation of proteins into the polymer

vesicles, a maximum of 25 monomers per vesicle is expected to be incorporated based on the

molecular weight of the vesicles estimated from static light scattering. Using the calculated

permeability of 74 m/s, 25 monomers per vesicle and the surface area of each vesicle, the

calculated water permeability for each AqpZ tetramer is 13 x 10-14 cm3/s, which is similar to that

reported for AqpZ reconstituted into liposomes (≥10 x 10-14, (Borgnia et al. 1999)).

Figure 2.2: Stopped flow light scattering experiments (a) Increase in relative light scattering with and without reconstituted AqpZ into the ABA polymer at 5.5 ° C at a molar ratio of 200:1 (polymer:protein). Fits are shown as guides. The initial rise rates were used to calculate the permeability. As seen from (a) a rise cannot be calculated for the pure ABA polymer vesicles (b) shows the rise in scattering between 2 and 10 s for the polymer that was used in calculating permeability at 5.5 ° C.

2.3.4 Calculation of Activation Energies

Activation energies of water transport calculated by conducting experiments over a wide

range of temperatures can be used to characterize the transport across the vesicle membranes as

30

diffusion-driven or channel-mediated. Calculated permeabilities for the AqpZ-ABA vesicles

indicate an increase of 38 to 94 times over ABA vesicles in these experiments. In Figure 2.3, the

exponential constants obtained from these experiments are plotted against the inverse of

temperature to determine the Arrhenius activation energies. The Arrhenius activation energy

calculated for the ABA vesicles was 8.7 kcal/mol, while the AqpZ-ABA vesicles had a value of

3.4 kcal/mol. The higher value obtained for ABA membranes is consistent with the values of

activation energies reported for polymer membranes in the measured temperature range

(Garybobo 1971; Mehdizadeh et al. 1989), indicating transport by diffusion through the polymer.

The low activation energy for the AqpZ-ABA vesicles is strong evidence for channel-mediated

water transport across the vesicle membrane. This value is also consistent with the low

activation energy values obtained with proteoliposomes with incorporated AqpZ (Borgnia et al.

1999) and with oocytes with inserted AQP1 (Preston et al. 1992).

Figure 2.3: Arrhenius plots for calculation of activation energy for osmotic transport of water across polymer membranes with incorporated AqpZ (ABA with AqpZ) and pure polymer membranes (ABA).

31

2.3.5 Reflection Coefficients

The reflection coefficient of solutes in membrane systems determines the extent to which a

particular solute will be excluded from the filtered water. The relative reflection coefficients of

salt, urea, glucose, and glycerol were determined using a procedure described by Meinild et al

(1998). Glucose was taken as the reference solute with an assumed reflection coefficient of 1

because of its relatively large molecular size. The calculated reflection coefficients of salt,

glycerol and urea were >1 indicating higher rejection of salt, urea and glycerol when compared

to glucose (data not shown). These reflection coefficients are higher than the values reported by

Meinild et al (1998) for the same solutes but are within the experimental error for these

calculations.

2.3.6 Effect of Protein-to-Polymer ratio on permeability

Stopped flow experiments were conducted with protein to polymer molar ratios of 1:25, 1:50

1:100, 1:200, 1:500 and 1:1000 at 5.5 ° C. A dramatic increase in the permeability followed by a

sharp decrease was observed with increasing concentration of protein as shown in Figure 2.4.

At a ratio of 1:50 the increase in permeability over pure polymer vesicles is approximately 3000

times.

Figure 2.4: Effect of increasing AqpZ concentrations on the permeability of ABA polymers (a) light scattering results normalized to fit between 0 and 1. Curves for different ratios have been offset for clarity and fits are shown as guides. Data at 0 AQP: ABA ratio shown at a fixed offset. (b) Increase in calculated permeability with increase in protein to polymer ratio.

32

2.4 DISCUSSION

Dramatic improvements in the efficiency of water treatment membranes may result from the

development of biomimetic membranes with high permeability and selectivity. In this work we

present the synthesis of a novel ABA triblock copolymer with incorporation of the bacterial

water channel protein- AqpZ, data on the low permeability of the ABA polymer alone, and the

large increase in permeability upon incorporation of AqpZ. This increased permeability varied

with the AqpZ:ABA ratio and showed an activation energy indicative of channel mediated

transport. Furthermore, the selectivity of the AqpZ-ABA membrane for water over small solutes

such as salt, glucose, urea and glycerol was demonstrated.

The dramatic increase in permeability observed upon incorporation of AqpZ indicates that the

water channel protein is functional in the synthetic context, as has been seen previously for other

membrane proteins in a similar polymer (Graff et al. 2002). The magnitude of increase in

permeability and the excellent solute rejection capabilities demonstrate the potential benefit of

such membranes for water treatment and drug delivery. The ABA triblock copolymer was found

to be relatively impermeable when compared to other membranes, with a productivity of 0.22

(m/s)bar-1 at 20°C as compared to the other reported value for a similar diblock copolymer

(Polyethylene oxide-Polyethylethylene designated OE-7) with normalized permeability of 5.7

(m/s)bar-1 at 20°C (calculated from Discher et al, 1999). The large difference in the size of the

hydrophobic blocks between the ABA polymer (110 units of PDMS) and the diblock copolymer

studied by Discher et al (37 units of Polyethyleneethylene (EE)) could account for this

difference. The permeabilities of lipid membranes are reported to be an order of magnitude

higher than OE-7 copolymer (Discher et al. 1999). The incorporation of AqpZ increases the

productivity of ABA membranes by ~800 times (Figure 2.5). Additionally, the productivitites of

AqpZ-ABA membranes reported here exceed those for any salt rejecting membrane. A

comparison of the measured permeability values to those measured by other researchers for

block copolymer membranes and to commercial reverse osmosis and forward osmosis

membranes is shown in Figure 2.5. Although these values were measured using different

procedures, it can be seen from this comparison that the protein-polymer system investigated

here represents more than an order-of- magnitude improvement over existing commercial

solute-rejecting membranes.

33

Figure 2.5: Comparison of reported permeability values for polymeric membranes to those obtained in the study. FO is commercial forward osmosis membrane with data from (McCutcheon and Elimelech 2006) at 20 ° C. RO is commercial reverse osmosis desalination membrane with data from Matsura (Matsura 2001) at room temperature (assumed 25 ° C) PB-PEO is Polybutadiene Polyethylene diblock polymer with data from Discher et al (1999) at temp of 20 ° C. ABA represents the polymer vesicles used in study with permeability calculated at 20 ° C. ABA+ AQP represents the polymer vesicles with incorporated AqpZ at 1:100 molar ratio used in this study at 20 ° C. Data for ABA and AqpZ were obtained at 5.5 ° C and calculated at 20 ° C using Ea values.

The AqpZ-ABA system studied here may benefit from further optimization of the protein

incorporation conditions. This will allow production of membranes with a greater enhancement

in permeability. Increasing the amount of AqpZ leads to a trend of increase in permeability

followed by a decrease similar to that seen with lipid membranes. In experiments with lipid

membranes a limiting concentration of AqpZ was observed around a weight ratio of 1:200

protein to polymer (Borgnia et al. 1999). We see such a limiting concentration at a ratio of 1:19

protein to polymer (1:50 molar ratio). The higher molecular weight of the polymer compared to

lipids might play a role in this difference. The limiting concentration observed is probably due

34

to the method of incorporation utilized. At the higher concentration of AqpZ a large amount of

detergent present in the stock solution of AqpZ was added to the reconstitution mixture leading

to a reconstitution mixture detergent concentration of ~ 0.4%. This may have resulted in a low

efficiency of reconstitution for AqpZ leading to lower permeabilities. Unfortunately, the number

of AqpZ inserted per vesicle have not been determined experimentally for inserted AqpZs in

previous studies (Borgnia et al. 1999) as well as this study to allow a definitive conclusion.

Additionally, other studies have shown that vesicles with a AqpZ to polymer weight ratio of 1:1

can be synthesized using slow detergent removal methods (Dolder et al. 1996; Scheuring et al.

1999). This indicates that higher permeabilities than that reported here might be possible with

other reconstitution methods. These results also indicate that membranes with specified levels of

water permeability can be synthesized by controlling the concentration of AqpZ – a property that

may be useful for drug delivery applications.

The protein polymer membranes examined in this study show excellent salt rejection and high

permeabilities ideal for water treatment membranes used for desalination. Excellent control

over the permeability of polymers was also demonstrated. Incorporation of AQPs or suitable

molecular mimics into compatible synthetic polymers such as the block copolymer system

investigated in this study is an innovative approach for making solute-rejecting membranes for

medical, industrial and municipal desalting applications.

2.5 MATERIALS AND METHODS

The detergent dodecyl maltoside used in protein purification was obtained from Anatrace

Chemicals (Maumee, OH, USA). Ni-NTA agarose beads were obtained from Qiagen,

Inc.(Valencia, CA, USA). Sepahrose 4B was used for chromatographic separation was obtained

from Sigma Aldrich (USA and Switzerland). Other chemicals were obtained as described in the

following sections.

2.5.1 Expression and Purification of AQP-Z

The AqpZ overexpression vector pTrc10His AqpZ was obtained from Dr. Peter Agre (Duke

University Medical Center, Durham, NC, USA) and transformed into the Escherichia coli strain

JM109 by elecroporation. Growth, isopropyl- D-thiogalactoside induction and Ni-NTA

affinity chromatography purification of AqpZ were performed as described in (Borgnia et al.

35

1999) with two modifications. The induction time was optimized at 7-8 hours and this was used

for all the protein production experiments. The ultracentrifugation setup for recovery of

membrane fractions was replaced by the use of a Biomax filter (MWCO of 30 KDa, Billerica,

MA,USA) in a centrifuge for 45-60 minutes for removal of smaller molecular weight material.

Protein purification was followed by solubilization with cracking buffer (Sambrook et al. 1989)

with 1% dodecyl maltoside and running polyacrylamide gel electrophoresis(Sambrook et al.

1989). The purified protein was quantified using the Lowry method (Lowry et al. 1951).

2.5.2 Synthesis of ABA polymer

The ABA polymer was a symmetric poly- (2-methyloxazoline)-block-poly (dimethylsiloxane)-

blockpoly (2-methyloxazoline) (PMOXA15-PDMS110-PMOXA10). The first step of the synthesis

procedure involved acid catalyzed polycondensation of dimethoxydimethyl silane in presence of

water and end-capper resulting in butylhydroxy terminated bi-functional PDMS. Liquid PDMS

was purified by vacuum stripping at 80°C and precipitation in equal (by weight) water/methanol

mixture. Purified PDMS was reacted with triflicacid anhydride in hexane at -10°C for three

hours resulting in triflate-PDMS bi-functional macroinitiator. The reaction mixture was than

filtered under argon through a G4 filter. Hexane was evaporated under vacuum and dry ethyl

acetate was added as reaction solvent. Addition of dry 2-methyl-2-oxazoline resulted in

symmetric ring-opening cationic polymerization of PMOXA blocks on the macroinitiator. The

reaction was terminated by addition of a methanol solution of potassium hydroxide. The

synthesis of a similar lower molecular weight polymer system is described in further detail in

Nardin et al, 2000.

2.5.3 Preparation of ABA vesicles and AQP incorporation

Block copolymer vesicles (1-2 wt. % of polymer in phosphate buffer saline (PBS) at pH 7.4)

were prepared using the film rehydration method. 60 mg of polymer was first dissolved in

chloroform (5-10 ml) and the chloroform was evaporated slowly in a rotary vacuum evaporator

at 40°C and a vacuum of less than 400 mbar to form a even film on the inside of round bottomed

flasks. This film was then further dried under high vacuum of 0.3 mbar for at least four hours.

10 ml PBS was then added drop wise to the film with alternating vigorous vortexing and periodic

sonication (of durations less than 30s) over several minutes. This mixture was then left stirring

36

for at least eight hours. This suspension was then extruded several times through a 0.4 micron

track etched filter (Isopore, Millipore Corporation, Billerca, MA,USA), followed by a 0.2 micron

track etched filter (Nucleopore, Whatman, Newton, MA, USA) to obtain monodisperse

unilamellar vesicles. For reconstitution experiments 500 L of an AqpZ stock solution (1.6 mg

mL-1 in 1.5% dodecyl maltoside, 66 mM KH2PO4, 133 mM NaCl, 13% glycerol, 3.33 mM

mercaptoethanol (BME), and 33.33 mM Tris was added during the formation of the polymer

vesicles and subsequent steps were completed on ice. The resulting protein-containing vesicles

were purified chromatographically using a column packed with Sepharose 4B to remove non-

incorporated protein and detergent traces.

2.5.4 Characterization of vesicles

Light Scattering: The static and dynamic light scattering experiments were performed according

to the description presented in Nardin et al, 2000 (Nardin et al. 2000) using a commercial

goniometer (ALV Langen) equipped with a frequency-doubled Nd:YAG laser (ADLAS,

wavelength 532 nm) at scattering angles between 30° and 150°.

AFM: AFM measurements were conducted using a Picoscan SPM LE scanning probe

microscope equipped with a Picoscan 2100 SPM controller (Agilent Technologies, Chandler,

AZ, USA). Measurements were conducted in tapping mode using a Si cantilever (NCH)

(Nanosensors, Neuchatel, Switzerland). The length of this cantilever was 125 m and the

nominal force constant was 42 N/m. Samples were prepared by placing a dilute vesicle

suspension on freshly cleaved mica for 1 minute and then carefully washing with double distilled

water.

TEM: Transmission Electron microscopy was conducted on vesicle samples using a Phillips 400

TEM. The samples were prepared by dilution up to 1000 times and then stained with 2% uranyl

acetate on plasma treated copper grids.

cryoTEM: The vesicle suspension was deposited on holey carbon grid and frozen rapidly by

plunging into liquid ethane above its freezing point using a cryo holder. This holder was then

transferred to the TEM and imaged in transmission mode at 200kV at liquid nitrogen

temperature. The equipment used for this measurement was a Zeiss 922 Omega microscope with

a Gatan CT3500 Cryo Holder and a Gatan Ultrascan 1000 camera system.

37

Permeability measurements: The permeability of the two types of vesicles were determined

using a stopped flow procedure (Borgnia et al. 1999). The permeability was measured by

following the light scattering of the preparation in a stopped-flow apparatus (SV.17MV, Applied

Photophysics, Letterhead, UK). The water permeability was measured by rapid mixing of the

vesicles with a solution containing the osmotic agent (salt, glucose, glycerol or urea) causing

water efflux from the vesicles. The changes in light scattering caused by this shrinkage were

recorded at an emission wavelength of 600 nm in the stopped flow apparatus. Under these

conditions a reduction in vesicle volume leads to an increase in the relative light scattering

measurements as described in Borgnia et al, 1999 according to the methods developed by Milon

et al, 1986. These experiments were conducted with pressure in the mixing cell held constant at

7 bar to minimize pressure variations that could complicate the analysis of kinetics. Results were

corrected for the mechanical compression resulting from this pressure by subtracting the baseline

observed in control experiments without osmotic agents. These data were then fitted to an

exponential rise equation to calculate the exponential coefficient and the osmotic water

permeability (Pf) was calculated using the following expression (Milon et al. 1986):

Where, k is the exponential rise rate constant for the initial rise in the light scattering curve, S is

the initial surface area of the vesicles, Vo is the initial volume of the vesicles, Vw is the molar

volume of water (18cm3), and Δosm is the difference in osmolarity driving the shrinkage of the

vesicles.

Several sets of experiments were conducted with similar conditions but different preparations

(prepared in different batches) to ensure repeatability. Additionally, initial experiments with up

to 12 traces per experimental condition showed that five traces were adequate for providing

sufficient data for analysis, so in subsequent detailed experiments a minimum of five were

acquired per experimental condition. Data analysis was conducted by averaging a minimum of

five traces for each scattering experiment and then fitting the initial rise to an exponential

equation using the curve fitting toolbox available through the MATLAB software (Mathworks,

Natick, Massachusetts, USA).

osmWof VVSkP )//(

38

Stopped flow experiments were repeated at different temperatures (5.5, 7.5, 10, 12.5 and 15 °C)

for the ABA and AqpZ-ABA vesicles. The exponential rise rates calculated from these

experiments exponential were plotted against the inverse of temperature to determine the

Arrhenius activation energies.

The reflection coefficients for salt, urea and glycerol were calculated based on the method

presented by Meinild et al, 1998 (Meinild et al. 1998). Glucose was used as the reference solute

and the comparative experiments were all conducted at 15°C.

39

3 BIOLOGICAL ROLE FOR BACTERIAL AQUAPORIN GATING DISCOVERED IN

BLOCK COPOLYMER MEMBRANES


PMOXA-PDMS-PMOXA polymer synthesis and transformation of the ptrcHis10AqpZ plasmid

in to E.coli.

The results described in this chapter have been submitted for publication.

3.1 ABSTRACT

Block copolymers can mimic lipids in structure and function, while being engineered for

desired chemical and mechanical properties. Use of a pH-insensitive, low-permeability triblock

polymer (Polymethyl oxazoline- Polydimethyl siloxane - Polymethyl oxazoline or PMOXA-

PDMS-PMOXA) as a matrix for reconstitution allowed discovery of pH gating of the bacterial

water channel protein Aquaporin Z (AqpZ). AqpZ was reversibly closed at pH ≤ 4 in polymer

vesicles and in whole cells of Escherichia coli. Furthermore, pH gating of AqpZ was important

for survival of acid and simultaneous acid and hypoosmotic shock, demonstrating the biological

relevance of this phenomenon. This work utilized a hybrid biological-synthetic system to provide

new insight into the physiological role of microbial aquaporins.

3.2 INTRODUCTION

Aquaporins mediate rapid and highly specific water transport across cell membranes

(Preston et al. 1992), and defects in aquaporins are associated with several human diseases (King

et al. 2000). In contrast, the role of aquaporins in microorganisms is uncertain. The large surface

area to volume ratios found in microorganisms should provide sufficient water transport via

passive diffusion without the need for a dedicated channel protein (Hill et al. 2004; Tanghe et al.

2006). Aquaporins are not essential in microorganisms, as evidenced by their incomplete

distribution (Zardoya 2005) and by the subtle effects observed in strains where the aquaporins

have been eliminated via inactivation of the aqp gene (Calamita et al. 1998; Soupene et al. 2002;

Hernandez-Castro et al. 2003; Shapiguzov et al. 2005). Indeed, some laboratory strains of yeast

40

appear to have gained a competitive advantage by inactivating their aquaporins (Bonhivers et al.

1998). Their existence, on the other hand, suggests some benefit. Proposed roles are diverse and

include involvement in osmoregulation, sporulation, cell expansion prior to cell division, and

freeze tolerance (reviewed by Tanghe et al (Tanghe et al. 2006) and Calamita (Calamita 2000)).

In plant and mammalian aquaporins, water transport is often regulated, including closure or

gating in response to changes in pH or osmolarity, and this regulation has provided insight into

the physiological roles of specific aquaporins (reviewed by Hedfalk et al (Hedfalk et al. 2006)).

Previous pH studies generally focused on the range between pH 6 and pH 8, perhaps due to

constraints imposed by pH effects on the liposome or oocyte systems used, and found a wide

range of effects. The plant plasma membrane intrinsic protein 2 (PIP2) subfamily of aquaporins

and the human AQP3 have reduced permeability at pH 6 or below pH 7, respectively (Tournaire-

Roux et al. 2003; Zelenina et al. 2003), while the mammalian AQP0 (Nemeth-Cahalan et al.

2004) and AQP6 (Yasui et al. 1999) have increased permeability at acidic pH, and mammalian

AQP4 & AQP5 are reported to be insensitive to pH (Zelenina et al. 2003). However, the

Escherichia coli aquaporin AqpZ is insensitive to two conditions affecting other aquaporins: 1

mM mercury and changes in pH between 5 and 7 (Borgnia et al. 1999), and to our knowledge no

experimental evidence of gating in a prokaryotic aquaporin has been reported prior to this work.

The structure and function of membrane proteins, including aquaporins, are typically

studied via reconstitution in a lipid matrix, but these investigations are limited by the lipid

properties. In particular, lipids have high permeability for water and gases, making it difficult to

study transport of these molecules (Verkman 2002; Uehlein et al. 2003; Hill et al. 2004).

Liposomes can also be strongly affected by pH, ionic strength, and temperature(Grit and

Crommelin 1993), complicating studies that need to be conducted under these conditions. To

investigate a broader range of membrane protein properties and as a platform for technology

development, we envision the increased use of synthetic block copolymer membranes with

tailorable properties.

Symmetric, ABA-type triblock copolymers composed of poly-(2-methyloxazoline)-poly-

(dimethylsiloxane)- poly-(2-methyloxazoline) (PMOXA-PDMS-PMOXA, referred to as ABA

throughout the rest of this paper) mimic lipid bilayers in their structure (Figure. 3.1) and ability

to form vesicular structures (Nardin et al. 2000). The feasibility of using ABA polymers for the

study of membrane proteins is supported by studies demonstrating insertion and activity of

membrane proteins, including AqpZ (Kumar et al. 2007). Specifically, AqpZ-rich ABA block

copolymer biomimetic membranes may have an important role in addressing world water supply

issues as alternative desalination membranes (Kumar et al. 2007). The use of PMOXA as the

hydrophilic block also provides the possibility of adding antifouling properties in this and other

applications (Konradi et al. 2008) where protein deposition is undesirable. The polymer vesicles

have several advantages over liposomes: i) they can be engineered to provide the desired

properties such as fluidity or morphology by the choice of blocks and block lengths (Discher and

Eisenberg 2002) or their length ratio (Zhang and Eisenberg 1995) ii) their end groups can be

modified (Nardin et al. 2000; Napoli et al. 2008) by molecules such as biotin (for recognition and

immobilization ) (Grzelakowski et al. 2009), methacrylate (for stabilization by crosslinking)

(Nardin et al. 2000), fluorescent molecules (for imaging) (Grzelakowski et al. 2009) and even

drugs (for drug delivery) (Peer et al. 2007) iii) they are highly stable (Discher et al. 1999), and

iv) they can be engineered to be sensitive (Sauer et al. 2001) or insensitive to pH changes (Figure

3.2). In this work, the use of pH-stable, low permeability ABA triblock copolymers permitted the

discovery of pH gating of AqpZ.

Poly‐2‐methyl‐2‐oxazoline Polydimethylsiloxane Poly‐2‐methyl‐2‐oxazoline

N NSi

O OSi Si

n OOHHO

O m m

Hydrophilic

Hydrophobic

Figure 3.1: Structure of PMOXA-PDMS-PMOXA polymer

41

Figure 3.2: AqpZ-ABA vesicles are stable over a wide pH range. To evaluate the pH stability of the AqpZ-ABA vesicles, dynamic light scattering measurements were conducted following overnight incubation at different pH. The results demonstrate the stability and uniform size of the vesicles between pH 2 and pH 12. Each point was calculated from 5 data points at different scattering angles. Error bars represent 95% Confidence Interval (C.I.) values.

3.3 RESULTS

3.3.1 Closure of AqpZ at low pH in polymer vesicles

During a systematic investigation of the permeability of ABA polymer vesicles with

incorporated AqpZ (AqpZ-ABA vesicles), we noticed that permeability decreased by 81%

between pH 6 and pH 4 (Figures 3.3 (a) and (b)). Two subsequent experiments suggested that

this was not due to damage of the polymer. First, sizing experiments suggested that the ABA-

AqpZ vesicles are stable at low pH (Figure 3.2). Second, the activation energies of water

transport for the AqpZ-ABA vesicles at pH values of 4, 7.2, and 12 are in the range of channel-

mediated transport (1.8 to 3.1 kcal/mole (Figure 3.4)) and lower than that through pure polymer

(8.7 kcal/mol (Kumar et al. 2007)), indicating that transport is still mediated by AqpZ throughout

this pH range. The permeability of AqpZ-ABA vesicles at pH 2 and 4 (84 and 107 m/s,

respectively), while lower than at neutral pH, is still higher than pure polymer vesicles (0.8

m/s(Kumar et al. 2007)), indicating that not all AqpZ activity is eliminated under these

conditions.

42

Figure 3.3: AqpZ-ABA vesicles have a lower permeability at acidic pH and this decrease is reversible. (a) Normalized light scattering response from a representative hyperosmotic shock experiment on AqpZ-ABA vesicles prepared at pH 4, 7.2, and 12. (b) A significant decrease in permeability (no overlap of 95% Confidence Interval (C.I.) error bars) is seen between pH 6 and 4. (c) AqpZ closure is reversible. AqpZ-ABA vesicles were tested at pH 4.0 (or 7.2), resuspended at pH 7.2 (or 4.0), and tested again. Error bars are 95% C.I. values from exponential fits of trace data (>8) for each condition.

Figure 3.4: Activation Energy for AqpZ-ABA vesicles at pH 4, pH 7 and pH 12. Based on permeability measurements at different temperatures, the activation energies of water transport at pH values of 4 (squares), 7.2 (circles), and 12 (triangles) are between 1.8 to 3.1 kcal/mole, indicating that transport is channel mediated.

To distinguish between damage and gating of AqpZ at low pH, the reversibility of this pH

effect was examined. The toughness of these vesicles allowed a batch of vesicles to be recovered

43

after stopped flow and reused at a different pH. Vesicles transferred from pH 4.0 to pH 7.2

regained 123% of the permeability of vesicles originally at pH 7.2 (Figure 3.3c). The recovery

following exposure to pH 2 was also complete (Figure 3.5). Thus, the low permeability observed

at acidic pH was fully reversible, indicating gating of AqpZ.

Figure 3.5: pH gating reversibility and control experiments for AqpZ-ABA vesicles. pH gating reversibility and control experiments shown for pH values between 7.2 and 2.0. Permeabilities were calculated from average of 8 or more stopped flow traces and error bars represent 95% C.I. values.

3.3.2 Closure of AqpZ at low pH in E. coli

This pH gating is an intrinsic property of AqpZ rather than an artifact of the synthetic

polymer environment, based on similar experiments with whole cells of E. coli (Figure 3.6a).

Comparing the permeabilities of strains with normal, overexpressed, and no aqpZ at pH 7.4, the

overexpression strain had the expected increase in permeability compared to the wild type strain.

The activation energy of water transport in the overexpression strain was 1.6 kcal/mol for pH 7.4

and 2.6 kcal/mol for pH 4 (Figure 3.7), indicating that the increased transport is still channel

44

mediated. With respect to pH, the wild type aqpZ strains (ME9062 and JM109 pTrc10His1) and

the aqpZ overexpression strain showed 15%, 44%, and 50% lower permeability, respectively, at

pH 4 than pH 7.4, while no decrease was observed in the aqpZ null strain. These results

demonstrate pH gating of AqpZ in E. coli.

Figure 3.6: Whole cells also exhibited an AqpZ-dependent, reversible decrease in permeability at low pH. (a) E. coli cells were less permeable at low pH. This decreased permeability was due to AqpZ, as an overexpression strain (AqpZ+) showed a larger decrease (50%) and no significant decrease was detected in an aqpZ null mutant (AqpZ-). Inset shows the less permeable strains at higher resolution. The results are averaged over 5 or more experimental traces. (b) Reversibility. Permeability of the overexpression strain recovered when the pH was changed from 4 to pH 7.4. Average permeabilities from three independent cultures are shown. Error bars represent 95 % C.I. values.

45

Figure 3.7: Activation Energy for E. coli water transport at pH 4 and pH 7. Permeability measurements were conducted at temperatures between 5 and 25C. The activation energies of water transport at pH values of 4 (circles) and 7.4 (squares) were calculated at 2.6 and 1.6 kcal/mol respectively.

As in the polymer vesicles, the observed decrease in the permeability of E. coli cells at pH

4 is not due to irreversible damage, because the original permeability is recovered upon

resuspension at neutral pH (Figure 2b). When the pH is increased from pH 4 to pH 7.4, the

resulting permeability of the overexpression strain is 93 % (95 % C.I. values, 74%, 115%) of its

original permeability at pH 7.4, indicating that the decrease in permeability at low pH is

reversible.

3.3.3 Effect of AqpZ on survival of acid and osmotic shock

While considering the possible physiological relevance of pH gating of AqpZ, we

investigated the effect of AqpZ on cell survival under separate and simultaneous acid and

osmotic shock (Figure 3.8). To avoid complications resulting from different growth rates in the

induced aqpZ overexpression strain and the other strains considered here, the overexpression

strain was only tested under uninduced conditions. However, even under uninduced conditions,

the permeability of the uninduced aqpZ overexpression strain is high, and at pH 4 its

46

permeability is higher than the wild type and null mutant under any conditions. In agreement

with the permeability data, this strain also has the most pronounced response to acid shock

(Figure 3.8 (b)). For osmotic shock alone, no significant difference in survival was observed

between the three strains, in agreement with previous reports indicating that AqpZ is not

essential for survival of osmotic shock (Calamita et al. 1998; Soupene et al. 2002). The

simultaneous acid and osmotic shock had the most pronounced effect, with all strains showing

lower survival than would be predicted based on separate shock experiments, and again with the

most permeable strain showing the lowest survival.

Figure 3.8. Lower permeability strains survive acid and simultaneous acid/hypoosmotic shock better. The acid shock was from pH 7.4 to 2.5 and the osmotic shock was from 300 to 100 mOsm (hypoosmotic), both for 30 minutes. All error bars represent 95 % C.I. values. At least three independent cultures were examined for each experimental condition.

3.4 DISCUSSION

This work demonstrates the biological relevance of a property discovered in a synthetic

membrane, illustrating the potential of hybrid biological-synthetic systems to contribute new

insight to biological systems. Our results demonstrate the reversible closure of AqpZ at pH 4 in

synthetic polymer vesicles and in whole cells. The low intrinsic permeability of these polymer

vesicles (Kumar et al. 2007) allowed sensitive determination of the change in AqpZ

permeability, and the insensitivity of the polymer to pH changes allowed investigation of a broad

range of pH conditions. The discovery of pH gating in the bacterial aquaporin AqpZ has

identified new directions for investigating the molecular mechanism of aquaporin gating and the

role of aquaporins in bacterial physiology.

47

The neutral pH permeabilities of our strains were consistent with previous reports using

different wild type (Mallo and Ashby 2006), null and overexpression strains (Hubert et al. 2005)

of E. coli. However, one apparent contradiction should be noted. Mallo and Ashby detected

<10% of wild type permeability in an aqpZ null mutant (Mallo and Ashby 2006), while our data

showed 84% of wild type permeability for another aqpZ null mutant. The reason for this

discrepancy has not been determined, but it could reflect differences in the strains or

experimental protocols.

The presence of pH-mediated AqpZ closure in both polymer vesicles and whole cells

indicates that pH has a direct effect on AqpZ. Detailed molecular mechanisms for pH gating

between pH 6 and pH 7 by plant and mammalian aquaporins have been proposed (Tournaire-

Roux et al. 2003; Zelenina et al. 2003; Nemeth-Cahalan et al. 2004; Törnroth-Horsefield et al.

2006). However, the molecular mechanism for the gating observed here is likely to be distinct

from those in plant and mammalian aqauporins, because the residues involved in these

mechanisms are not conserved in AqpZ (Figure 3.9). The pH range suggests the involvement of

a histidine residue, and the experimental assays used in this work provide a basis for

investigating the molecular mechanism.

Figure 3.9: Residues involved in pH gating in plant and mammalian aquaporins are not conserved in bacterial aquaporins. Aquaporin amino acid alignments are shown for regions identified in the literature as important for pH gating. The first segment corresponds to Loop A, the second and third to Loop C, and the fourth segment is from Loop D. Specific residues that affect pH gating are circled in the figure and include: H40 for hAQP0 (Nemeth-Cahalan et al. 2004); H53, Y124, S152 and H154 for hAQP3 (Zelenina et al. 2003); H193 and L197 for SoPIP 2;1 (Zelenina et al. 2003) and H197 and L201 for PIP 2;2 (Tournaire-Roux et al. 2003). Amino acid sequences were retrieved from the Genbank database (http://www.ncbi.nlm.nih.gov/) with the sequence for hAQP3 obtained from Uniprot database (http://www.uniprot.org/). The alignment was conducted using the Clustal W program (http://align.genome.jp/) and results are displayed using Cinema 5 (http://utopia.cs.manchester.ac.uk/cinema).

48

http://www.ncbi.nlm.nih.gov/

http://www.uniprot.org/

http://align.genome.jp/

http://utopia.cs.manchester.ac.uk/cinema

To consider the physiological relevance of pH gating of AqpZ, we developed a model

integrating pH gating of AqpZ with existing information on bacterial responses to osmotic and

acid shock (Figure 3.10). When exposed to hypoosmotic shock, aquaporins may provide a rapid

equilibration mechanism, while the increased pressure (and accompanying membrane stretching)

resulting from hypoosmotic shock activates mechanosensitive channels (Berrier et al. 1992;

Levina et al. 1999) to release small solutes. This prevents cells from bursting when a sudden

downshift in osmotic pressure occurs. At low pH, an increase in activation pressure of the

mechanosensitive channel MscL has been demonstrated (Kloda et al. 2006) and serves to prevent

acidification of the cytoplasm. However, this regulation also reduces the protective action of

MscL. The ability of cells to avoid a rapid influx of water, via pH gating of AqpZ, might

therefore be an important protective step under combined acid and hypoosmotic shock. This

model is supported by the reduced survival of more permeable strains under acid and combined

acid/osmotic shock (Figure 3.8).

Figure 3.10: Model for coordinated activity of aquaporins and mechanosensitive channels under pH and osmotic downshift conditions. Upon hypoosmotic shock aquaporins mediate rapid water transport into the cell, leading to rapid swelling. To prevent cells from bursting, mechanosensitive channels open and allow small solutes to be released. Under acid shock, mechanosensitive channel activation is lowered. The cell is therefore especially vulnerable to combined acid and hypoosmotic shock, unless water permeability of AqpZ is also reduced under acidic conditions.

49

While many questions remain about the physiological role of AqpZ, this report suggests a

new direction for such investigations, namely acid stress. Acid stress response also provides a

potential explanation for the apparent preferential distribution of AQP homologs in Gram

negative bacteria (Zardoya 2005), as the acid response in these organisms relies in part on

periplasmic acidification, while the Gram positive bacteria utilize different protective

mechanisms (Foster 2000). The acid and combined acid and osmotic shock conditions

considered here for whole cells are environmentally relevant, similar to conditions E. coli and

other pathogens might be exposed to upon passage through a host digestive system, and therefore

insights into bacterial survival under these conditions may also have relevance to public health

and food safety.(Diez-Gonzalez et al. 1998)

This work demonstrates that specifically designed biomimetic polymeric materials can

facilitate discovery of biologically relevant fundamental properties in hybrid synthetic-biological

systems. The robust block copolymer membrane platform provides the flexibility to conduct

assays of membrane protein properties under conditions that were previously limited by the

mechanical and chemical properties of lipid membranes.


3.5.1 AqpZ-ABA vesicles.

AqpZ, poly (2-methyloxazoline)-block-poly (dimethylsiloxane)-block-poly (2-

methyloxazoline) (PMOXA15-PDMS110-PMOXA15) polymer, and protein-polymer vesicles were

prepared as described previously (Kumar et al. 2007) using a protein to polymer molar ratio of

1:100 and adjusting pH with HCl or NaOH. Vesicles were purified using Sepharose 2B. Size and

stability of AqpZ-ABA vesicles synthesized at different pH values were evaluated before and

after overnight storage using dynamic light scattering as previously described (Kumar et al.

2007).

3.5.2 Vesicle permeability.

Permeabilities were calculated from stopped flow light scattering results(Kumar et al.

2007) with 0.85 M NaCl as the osmotic agent for polymer vesicles. Three sets of experiments

were conducted with similar conditions but independent preparations to ensure repeatability. A

minimum of eight traces were averaged for each experimental condition. Reversibility

50

experiments were conducted by collecting the vesicles after stopped flow, transferring them to

the second pH by repeated washes in a centrifugal filter (20kDa, Amicon Ultra 15, Millipore),

and repeating the stopped flow experiment.

3.5.3 Bacterial strains.

Four strains were used for the experiments reported here, an aqpZ null mutant, an aqpZ

overexpression strain, and their respective parents. The wild type E. coli strain was the parent

strain used for construction of the Keio collection (BW25113) as obtained from the National

BioResource Project (Japan) under strain number ME9062. Its genotype is rrnB lacZ4787

hsdR514 (araBAD)567 (rhaBAD)568 rph-1. The aqpZ null mutant (JW0859) was constructed

in ME9062 as part of the Keio collection and contains a kanamycin resistance gene inserted in

aqpZ (Baba et al. 2006). The AqpZ overexpression vector pTrc10HisAqpZ was obtained from

Dr. Peter Agre (Duke University Medical Center, Durham, NC, USA) and transformed into E.

coli strain JM109 (endA1 recA1 gyrA96 thi hsdR17 (rK-, mK

+) relA1 supE44 Δ(lac-proAB) [F'

traD36 proAB lacIqZΔM15]) by electroporation. To construct an isogenic control for the

overexpression strain, AqpZ was deleted from the pTrc10HisAqpZ plasmid using a EcoRI and

Pst1 double digest, overhangs were filled in with T4 DNA polymerase, and the resulting DNA

(pTrc10His1) was blunt end ligated and introduced into JM109 by electroporation.

3.5.4 Whole Cell Permeability Experiments.

These experiments were conducted using a stopped flow apparatus as described in Mallo and

Ashby (Mallo and Ashby 2006). All strains were grown overnight (15 hours) with shaking at 37

deg C in LB medium. 50 g/ml ampicillin was included as needed to maintain plasmids. The

cultures were then subcultured (1:100) into fresh LB medium. For induction of AqpZ, IPTG was

added 4 hours after subculturing. Eight hours after subculturing, the cells were harvested by

centrifugation at 10,000 g for 5 minutes at 4 deg C. The cell pellet was washed three times with

a solution of 100 mM PBS, with the addition of 50 g/ml kanamycin (or chloramphenicol for the

aqpZ null strain) to suppress protein synthesis during sample preparation and stopped flow

analysis. After washing, the cells were resuspended in a variable volume of PBS – antibiotic

solution at pH 7.4 or 4.0 to obtain a solution with a fixed transmittance (701%) and tested

within an hour.

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http://www.shigen.nig.ac.jp/ecoli/pec/gatewayAction.do?chromosomalMarkerId=773

Stopped flow experiments were conducted on the above cell suspensions with 1 M proline (also

adjusted to pH 7.4 or 4.0) as the osmotic agent and time bases between 0.2 and 5 s. The initial

volume (Vo) and surface area (S) of the cells were calculated as described in Mallo and Ashby

(Mallo and Ashby 2006) by assuming a length of 5 m and a diameter of 1 m, average values

for K-12 strains of E. coli under these growth conditions (Van De Merwe et al. 2004). Simple

exponential equations were used to determine the initial rise rate using the curve fitting toolbox

in MATLAB. The fit values were also tested using the curve fitting software provided with the

Applied Photophysics stopped flow for one set of experiments.

The reversibility of permeability change between pH values was tested as follows. An induced

culture of the overexpression strain was resuspended in pH 7.4 (or 4) buffer and part of this

culture was used to measure permeability. The remaining culture was spun down and transferred

to the second pH. Thus different aliquots of the same culture were tested at both pH values.

Five or more traces were obtained for each condition. At least three independent cultures were

tested.

3.5.5 E. coli survival experiments.

The experiments were conducted using a procedure similar to that described by Levina et al

(Levina et al. 1999). Single colonies from fresh overnight plates were grown overnight (12-15

hours) with shaking at 37 deg C in LB medium. They were subcultured 1:50 (for two sets of

experiments with AqpZ- and ME9062 experiments) or 1:100 (all other experiments) into fresh

LB medium supplemented with 0.2 M NaCl and grown for 4 hours. These cultures were diluted

1:10 (for experiments under both acid and osmotic shock conditions) or 1:100 (all other

experiments) into different media preparations to provide the desired shock. For control

experiments, dilution was performed in the same medium, while the acid shocks were performed

in LB at pH 2.5 and the osmotic shocks were in LB without supplemental NaCl. After 30

minutes of incubation at 37 deg C, serial dilutions were spread on LB agar plates and incubated

at 37 deg C overnight. Survival was calculated based on results of the control plates for each

culture.

52

4 SELF-ASSEMBLY OF MEMBRANE PROTEINS IN BLOCK POLYMERS


PMOXA-PDMS-PMOXA and NTA-polymer synthesis, transformation of the ptrcHis10AqpZ

plasmid in to E.coli, surface pressure measurements (Figure , and Brewster Angle Microscopy

measurements.

4.1 ABSTRACT

Incorporation of membrane proteins at high densities in lipid membranes to produce 2D

crystals is an excellent but challenging technique to study membrane protein structure in the lipid

environment (Jap et al. 1992). Such arrays could also have relevance to applications where a

large concentration of membrane proteins is beneficial. Increased sensitivity in sensor-based

applications and transport in transport-oriented applications are examples of such applications.

In this work we show evidence for crystallization of membrane proteins in a biomimetic triblock

copolymer. A templating diblock copolymer film with a nickel functionalized hydrophilic block

was first assembled at the air water interface. A ternary mixture of triblock copolymer, detergent

and histidine-tagged membrane protein was then added to the subphase. Slow detergent removal

using polymeric adsorbents led to formation of flat protein rich areas up to 1 micron in length.

Electron microscopy imaging and electron diffraction show that some of these areas are highly

ordered (crystalline). In addition to protein-polymer crystals that look similar to previously

described, lipid-based AqpZ 2D crystals and display loose ordering, strongly diffracting

structures were also observed. These structures may be pure block copolymer crystals or novel

2D crystals of block copolymers and AqpZ. The structures observed and the electron diffraction

results for these crystals are different from lipid-based 2D crystals.

4.2 INTRODUCTION

Membrane proteins provide specific and elegant transport mechanisms for water, ions and

nutrients needed by a cell to survive. They are also the cell’s sensor for environmental

conditions that range from presence of specific toxins to change in pH. Thus, understanding the

structure of membrane proteins is fundamentally important. In addition, incorporating these

proteins into cell membrane analogs could provide materials with targeted applications in water

53

purification (Kumar et al. 2007), enzymatic reactions (Astier et al. 2005), and contaminant

recognition, transport, and sensors (Gu et al. 1999; Braha et al. 2000; Nielsen 2009).

Incorporation of membrane proteins at high densities in lipid membranes to produce 2D crystals

is widely used to study membrane protein structure in the lipid environment (Jap et al. 1992).

Block copolymer membranes mimic the architecture of natural lipid cell membranes and allow

incorporation of functional proteins across the membrane (Taubert et al. 2004). However, in

contrast to lipid-based membranes, block copolymers can be tailored to have the stability and

toughness attributable to polymeric materials (Discher et al. 1999) and hence are more suitable to

formation of membrane protein based devices and applications.

Here we provide the first report on the possible formation of 2D membrane protein crystals

using amphiphilic block copolymers. The bacterial water channel protein Aquaporin Z was used

for these crystallization experiments. The procedure used was a monolayer based templating

procedure described previously for protein crystallization (Uzgiris and Kornberg 1983; Lévy et

al. 1999). However, instead of the commonly used nickel chelated lipid monolayer, a highly

detergent-resistant monolayer consisting of a nickel (Ni2+) functionalized Polybutadiene-

Polyethylene Oxide diblock copolymer (Nehring et al. 2009) was used. Additionally the 2D

crystal was assembled in a PMOXA-PDMS-PMOXA based triblock copolymer instead of the

natural or synthetic lipids used previously.

4.3 RESULTS

4.3.1 Monolayer formation

Langmuir trough film compression studies revealed transition between the dehydration of

the hydrophilic blocks of the diblock copolymer to the self assembly of the hydrophobic

polybutadiene blocks at around a mean molecular area of 100 Angstrom2 (Figure 4.1). Brewster

Angle microscopy of this self assembly process using the precursor hydroxyl terminated

hydrophilic block polymer (PB-PEO-OH) reveled that large areas (>100 microns) of stable

monolayer areas could be formed.

54

Figure 4.1: Film compression data for the Ni NTA parent diblock copolymer at the air water interface.

4.3.2 Templating AqpZ particles

Histidine tagged AqpZ molecules could be templated onto the NTA diblock copolyment

monolayer. Figure 4.2 shows a comparison between single particle images of AqpZ (panel a)

and templated AqpZ particles on the NTA polymer at different coverages (panels b and c) when

the monolayer was incubated with 40 g/ml protein. As seen from this figure both regularly

distributed templated proteins (Figure 4.2 (b)) as well as areas with aggregated proteins (Figure

4.2 (c)) are seen. This shows that templating of His-tagged membrane protein is possible using

this interfacial film.

Figure 4.2: Templating of AqpZ tetramers on Ni NTA block copolymer film. a) Single particle AqpZ micrograph showing AqpZ tetramers, negatively stained with 1% uranyl acetate. Scale bar 0.1 micron. b) Templated AqpZ molecules on the Ni NTA block copolymer film (Scale bar

55

0.2 micron) c) Higher density of templated AqpZ molecules showing aggregated areas. (Scale bar 0.2 micron)

4.3.3 Self assembly of protein-lipid films on templating layer

PC/PS lipids formed large micron size folded sheets at a LPR of 2 with a number of

wrinkled vesicles also seen. At lower LPR values vesicles and smaller flat sheets were observed.

Figure 4.3(a) and (b) show folded sheets and vesicles with bright edges indicating the presence

of packed proteins. The use of DOPC/POPC lipids show formation of flat single or

multilayered layered crystals at a LPR of 2 as shown in Figure 4.3(c) and 4.3(d). This result

was expected as POPC/DOPC lipids have been used for 2 D crystallization of AqpZ using other

(non-templating) methods (Ringler et al. 1999)

Figure 4.3: 2 D crystals of AqpZ formed in lipid using templating block copolymers. a) Large folded sheet formed at a LPR of 2 with PC/PS lipids. Scale bar 0.5 micron b) Wrinkled vesicles formed at a LPR of 2 with PC/PS lipids. Scale bar 0.5 microns. c) and d) Flat sheets formed at a LPR of 2 with DOPC/POPC lipids. Scale bar 0.2 microns.

4.3.4 Self assembly of protein-polymer films on templating layer

Three common structures were formed with an ABA polymer to protein ratio of four.

Figure 4.4(a) shows that large vesicles that were also formed under these conditions but were

56

apparently unordered as seen from a lack of diffraction spots in the fourier transform of their

image. The structures shown in Figure 4.4(b) seem to be crystalline. This is seen from the

power spectrum of the raw images (inset in Figure 4.4(b)) which reveals that this structure has a

tetragonal structure as would be expected from a AqpZ 2D crystal. The weak spots seen here

indicate that the crystal is weekly ordered. Figure 4.4(c) shows large flat-plate like structures, but

the power spectrum of the image by itself does not show diffraction spots. Of the above three

types of structures, only the structures shown in Figure 4.4(b) has the behavior expected of lipid-

based membrane protein 2D crystals, with diffraction spots occurring in the image power

spectrum at 40,000-50,000 magnification.

Figure 4.4: Representative structures formed using the monolayer technique. (a) Small ordered areas formed at a polymer to protein ratio of four. Inset shows power spectrum of the image showing spots indicating a tetragonal crystal. Scale bar 50 nm (b) large vesicles. Scale bar 100 nm. (c) Large flat plate like structures. Scale bar 200 nm.

However, as shown in Figure 4.5, direct electron diffraction of the flat plate like

structures commonly seen in such samples diffract electron quite strongly (4th order). This is the

limit of what could be expected to be resolved using a negatively stained membrane protein

crystal. However, these diffraction patterns are unexpected as AqpZ crystals in lipids rarely

show such bright diffraction spots under negative stain conditions. One clue to understanding

the origin of these spots is to determine the lattice spacing indicated calculated from these

diffraction patterns.

57

Figure 4.5: 2 D crystals formed in PMOXA PDMS PMOXA triblock copolymer (a) Large sheet formed at a polymer to protein ratio of four with PMOXA PDMA PMOXA polymers. Scale bar 0.2 micron (b) Close up of a) Scale bar 50 nm. (c) Electron diffraction of crystal shown in a using selected area diffraction.

4.4 DISCUSSION

This work was focused on synthesizing protein-rich polymer films. Such films were

formed at the scale of ~ 1 micron. Specifically, there were two significant findings of this work

that are discussed in the following paragraphs: a) Formation of a stable Ni2+-functionalized,

polymer monolayer at the air water interface which was resistant to detergent (up to 1% DDM),

which represents 180 times the critical micelle concentration, and b) Formation of 2D crystals of

AqpZ in block copolymers.

Stable functionalized diblock co-polymer monolayers such as those used in this study

could have two direct applications. The first application is for immobilizing proteins from

solutions. In particular, the high detergent resistance of this film is important to applications such

as the Ni-chelating lipid-based monolayer techniques proposed by Kelly et al (Kelly et al. 2008)

and an extension of this technique, the so-called “affinity grid”. In the affinity grid technique Ni-

chelating lipids are deposited onto a TEM grid, dried and stored (Kelly et al. 2008a; Kelly et al.

2008b). Both the monolayer and the affinity grid techniques have been suggested for extracting

specifically tagged proteins from crude cell lysate for direct three dimensional cryogenic electron

microscopy-based (cryo-EM) tomography and structure determination (Kelly et al. 2008). These

templating di-block co-polymers could be an excellent replacement for chelating lipids for such

applications, particularly as high concentrations of detergent are sometimes necessary for

solubilization of membrane proteins. The higher molecular weight of this polymer indicates, in

addition to a higher detergent resistance, higher mechanical and chemical stability. For the

“affinity grid” loss of the lipid monolayer to glycerol and/or detergents limits some of the

58

functionality of this method (Kelly et al. 2008a). Additionally, the hydrophilic block of the

diblock copolymer could be functionalized in a variety of ways in order to provide specific

recognition for proteins and other macromolecules. Here, the hydrophilic block was

functionalized with nickel providing recognition for histidine tagged proteins or possibly with

proteins with surface histidines (Pack et al. 1997). Functionalizing the hydrophilic end with

other affinity tags could provide a customized templating procedure. Secondly, the block

copolymer monolayer could be successfully used for 2D crystallization in lipids. 2D

crystallization of AqpZ using a monolayer technique has not been shown before. Here we tested

both a PC/PS based system as well as a previously used POPC/DOPC system. This versatile

diblock polymer system could be used in place of oxidation sensitive lipid based Ni-chelating

monolayers in 2D crystallization studies.

2D crystallization of AqpZ in block copolymers is the most significant finding of this

chapter. The crystals formed had a similar morphology to lipid-based AqpZ 2D crystals. The

quality of the crystals obtained thus far is not adequate for further processing. Additional efforts

should be directed towards making larger crystals, using other methods including dialysis, if

final structure of the protein in block copolymers is to be determined. Alternative methods of

transfer to the TEM grid and better imaging techniques including the use of cryogenic and low-

dose electron microscopy techniques under various tilt conditions should be utilized.

Nevertheless, this is a highly promising initial finding. The ability to form 2D crystals opens the

door to structure determination of membrane proteins in these polymers. Accurate structure

determination of proteins in polymers can provide a molecular basis for anomalous effects seen

after incorporation of membrane proteins into block copolymers and provides a basis for design

of novel functional materials. Thus, attempts should be made to from 2D crystals of such

proteins where a large change in conformation is suspected from activity measurements, and that

are of interest for applications. Moreover, a clear understanding of this phenomenon will

provide us with the tools to manipulate the extent of hydrophobic mismatch to control the level

of protein activity.

The presence of highly diffracting, large flat areas that do not seem to be similar to

previously described lipid-based AqpZ crystals was surprising. To better characterize these

areas, the electron diffraction pattern shown in Figure 4.6(b) was used to calculate the lattice d-

spacing values for the crystal shown in Figure 4.6(a). For lipid-based AqpZ crystals the d

59

spacing should be about 10 nm, as the unit cell size is in the range of a=b=10 nm. However, for

these flat areas the d spacing was quite large (~ 28 (100) and 20 nm (110)) indicating that this is

not a typical protein crystal. The possibility of this being an artifact from the staining process

was eliminated, as similar diffracting structures were found in unstained, cryo-plunged samples.

Another common cause of artifacts in TEM sample, parts of the carbon support, could also be

eliminated as a potential artifact as these are amorphous materials and would not show sharp

diffraction spots. These areas could be either pure block copolymer crystals or a novel form of

block copolymer – AqpZ 2D crystal. If these are pure block copolymer crystals, it would be the

first report of PDMS block copolymer crystallization though self assembly in an aqueous

solution and would be applicable to determination of the polymer structure. If these are novel

AqpZ-ABA block copolymer crystals it could be studied further to confirm the composition and

structure of these unique materials, particularly if AqpZ can be shown to be active.

Figure 4.6: Image (a) and selected area diffraction pattern (b) of a flat crystalline structures seen during crystallization at a polymer to protein ratio of four using a nickel templating monolayer.


The lipids used in this study were obtained from Avanti Polar Lipids, Alabaster, AL, USA.

The detergents dodecyl maltoside (DDM) and Octyl glucoside (OG) used in protein purification

were obtained from Anatrace Chemicals (Maumee, OH, USA). Ni-NTA agarose beads were

obtained from Qiagen, Inc. (Valencia, CA, USA). Transmission electron microscopy grids

were obtained from Ted Pella, Redding, CA, USA (Cu grids) or Pacific Grid-Tech, San

60

Francisco, CA, USA (Mo grids). N,N-Bis[(tert-butyloxycarbonyl)methyl]-L-lysine tert-

Butylester (Lys-NTA) was a gift from Prof. Robert Tampé’s group, Institute of Biochemistry,

Biocenter, Goethe Universität, Frankfurt/ Main and was used as received. Other chemicals and

supplies were obtained as described in the following sections.

4.5.1 Nickel functionalized diblock copolymer synthesis

This synthesis has been described in detail in a recent report (Nehring et al. 2009) and

therefore is described only briefly here. The polybutadiene-polyethyleneoxide diblock copolymer

(PB39-PEO36-O-) was synthesized via anionic polymerization. Poly (ethylene oxide)

polymerization was initiated by adding ethylene oxide to the living PB anions. After activation

with dicyclohexylcarbodiimide the polymeric carboxylic acid PB39-PEO36-SA-OH was reacted

with Lys-NTA. After deprotection and further purification, the yield of the functionalized block

copolymer was found to be 35%. To obtain the metal-polymer complex, the block copolymers

were stirred with a Nickel Chloride solution overnight until a green polymer solution was

obtained. This polymer is referred to as NTA polymer throughout this paper.

4.5.2 Triblock copolymer synthesis

The Polymethyloxazoline-Polydimethylsiloxane (PMOXA12PDMS55PMOXA12) block-

copolymer was synthesized by cationic ring-opening polymerization of 2-methyloxazoline using

a bi-triflate activated polydimethylsiloxane macroinitiator as described previously. The synthetic

route followed the one originally described in Nardin et al (Nardin et al. 2000). The molecular

weight of the polymer obtained from 1H NMR was 6325g/mol.

4.5.3 Membrane protein purification

Growth, isopropyl--D-thiogalactoside (IPTG) induction and Ni-NTA affinity

chromatography purification of histidine-tagged AqpZ were performed as described in Borgnia et

al (1999) with modifications described in Kumar et al (2007). The purified protein was

quantified using the Pierce 660-nm assay (Pierce Biochemical, Rockford, IL, USA). The

purified protein samples were dialyzed against elution buffer without imidazole prior to

experiments.

4.5.4 Monolayer formation studies

61

To determine appropriate conditions for formation of a stable diblock copolymer

monolayer at the air-water interface, film compression and Brewster Angle Microscopy (BAM)

techniques were used. Surface pressure-area (π-A) isotherms were obtained by using either a

Langmuir-Blodgett minitrough (surface area 273 cm2) or a BAM trough (surface area 432 cm2).

Both troughs were obtained from KSV Instruments Ltd., Helsinki, Finland. Prior to experiments,

the troughs were thoroughly cleaned with chloroform and ethanol, rinsed with double distilled

water, and filled with the aqueous subphase (either pure water or a phosphate buffer saline, 50

mM Na2HPO4, 10 mM KCl (Fluka AG, Buchs, Switzerland)), pH 7.4. The barriers were cleaned

with ethanol and rinsed with water. The Wilhelmy plate used in surface pressure measurements

was made of chromatography paper (ashless Whatman #1) with a perimeter of 20 mm. It was

equilibrated for at least 20 min. The surface was cleaned repeatedly through compression-

aspiration-expansion cycles and checked for impurities. The NTA polymer was spread dropwise

from chloroform solution (c = 1 mg/mL) on the air/water interface. The solvent was allowed to

evaporate for 10 min, and the monolayers were compressed at the rate of 10 mm/min. After each

measurement, the surface was cleaned and checked for impurities. All experiments were

performed at 20°C in a dust-free room, with the trough housed in a Plexiglas cabinet.

4.5.5 2 D crystallization studies

2D crystallization experiments were conducted in microtroughs built into Teflon block as

described in Lévy et al (Lévy et al. 1999). Each block had eight cylindrical troughs with a

diameter of 4 mm. Each well had a side port for addition of solutions and the overall volume

was approximately 60 microL. Citrate buffer (20 mM) containing 200 mM NaCl, 100 mM

MgCl2, 3 mM NaN3 and 10% glycerol at a pH of 7 was used for all the crystallization

experiments. A schematic of this process is provided in Figure 4.7. 60 L of buffer was first

added to each well to form a surface with a flat meniscus. To this 0.5 L of the templating

polymer dissolved in chloroform was added, targeting more than one but less than 2 monolayers

at a surface pressure equivalent to a mean molecular area of approximately 100 Å2 (determined

from Langmuir isotherm studies). This was equivalent to a concentration of 0.28 mg/ ml of

polymer in chloroform added to the surface of each cylindrical trough. A detailed calculation is

provided in Appendix B. The chloroform was left to evaporate for an hour.

62

A ternary mixture of ABA triblock copolymer, the detergent dodecyl maltoside (DDM),

and AqpZ protein was prepared and equilibrated overnight at 4 deg C. This mixture was then

added to the subphase of this monolayer. This mixture was designed to target a concentration of

100 g/mL of protein in the subphase. Polymer to protein ratios of between 0.2 and 4 were

screened and a 10% detergent (DDM) was necessary to solubilize ABA block copolymers. For

experiments with lipids, lipid to protein ratios of between 0.2 and 2 were screened. A 80%:20%

chicken egg phosphatidylcholine (PC)/ porcine brain phosphatidyl serine (PS) mixture and a

50%:50% 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/ 1-palmitoyl-2-oleoyl-sn-glycero-

3-phosphocholine (POPC) mixture was used for lipid experiments at lipid to protein ratios

between 0.2 to 2. A detergent concentration of 2% DDM was used to solubilize the lipids. After

addition of the ternary mixture the system was allowed to rest for 3-4 hours to allow binding of

the ternary micelles to the Ni2+ groups on the monolayer. Following this stabilization period

absorbent polymeric beads (Biobeads SM2) were added to the subphase at a dose of ten times the

detergent amount in the subphase, divided into equal batches of 6-8 additions over a 24-48 hour

period. Detergent removal drives self assembly of the protein polymer mixture.

water

NTA

PB-PEO-Ni NTA copolymer (in trough)

air

PB

PEO

Mixed Detergent, AqpZ, and

ABAmicelles

Slow detergent r

emoval

(Biobeads)

Ni2+Ni2+ Ni2+ Ni2+

airwater

Ni2+

Inject in subphase

Aquaporin Z

His tagHis tag

His tag

Dodecyl Maltoside(Detergent)

+

PMOXA-PDMS-PMOXA (block polymer)

PMOXA

PDMS

PMOXA

(a)

(b)

(c)

(d)

(e)

AqpZ 2D Crystalin block copolymer

NTA

Ni2+

NTA

Ni2+

NTA

Ni2+

water

NTA

PB-PEO-Ni NTA copolymer (in trough)

air

PB

PEO


ABAmicelles


ABAmicelles


ABAmicelles

Slow detergent r

emoval

(Biobeads)

Ni2+Ni2+ Ni2+ Ni2+

airwater

Ni2+Ni2+ Ni2+ Ni2+ Ni2+Ni2+ Ni2+ Ni2+

airwater

Ni2+

Inject in subphase

Aquaporin Z

His tagHis tag

His tag

Aquaporin Z

His tagHis tag

His tag

Aquaporin Z

His tagHis tag

His tag

Aquaporin Z

His tagHis tag

His tag

Dodecyl Maltoside(Detergent)

+


PMOXA

PDMS

PMOXA

Dodecyl Maltoside(Detergent)Dodecyl Maltoside(Detergent)

+


PMOXA

PDMS

PMOXA


PMOXA

PDMS

PMOXA

(a)

(b)

(c)

(d)

(e)

AqpZ 2D Crystalin block copolymer

NTA

Ni2+

NTA

Ni2+

NTA

Ni2+

Figure 4.7: Overall scheme for 2D crystallization of AqpZ in triblock copolymer using templating Ni2+ functionalized diblock copolymers.

63

The 2D crystals attached to the templating film were transferred onto non-glow

discharged Cu or Mo grids with thin carbon support for electron microscopy and negatively

stained with 1% uranyl acetate. Experiments were also performed with 40 g/ml AqpZ only (no

polymer or lipid) added to the monolayer subphase for comparison.

4.5.6 Electron Microscopy and electron diffraction

Electron microscopy was conducted on either a Phillips CM200 microscope at 120 keV

or on a JOEL 2100 microscope at 200 keV, both with LaB6 emitters. A liquid nitrogen cooled

Gatan Instruments Cryo-stage was used for cryo-electron microscopy with a FEI vitrobot used

for preparing hydrated frozen samples.

Single particle and templated AqpZ particles. AqpZ stock solutions, diluted to 25 microg/ml in

protein buffer, were applied to glow discharged copper grids and stained with 1% uranyl acetate

to visualize single AqpZ particles. AqpZ templated on monolayer (subphase concentration of ~

40 g/ml) was transferred onto non-glow discharged Cu grids and negative stained using 1%

uranyl acetate.

Electron microscopy of 2D Crystals and electron diffraction. 2D crystals were transferred onto

grids by depositing grids directly onto the monolayer for 2 minutes. The grids were then washed

twice with double disitilled water and stained with 1% uranyl acetate. Electron diffraction of

selected areas was recorded using Selected Area Diffraction apertures to select appropriate areas

or using the nanobeam diffraction mode for smaller diffracting areas.

Cryo-electron microscopy. In order to obtain images that were free from staining artifacts and

that may be suitable for further image processing 2D crystal solutions were embedded in a 2%

glucose or trehalose solution and loaded into the electron microscope using a liquid nitrogen

cooled cryo-stage. Electron diffraction patterns at low temperatures were recorded using a CCD

camera at appropriate exposure times to resolve the maximum number of spots possible.

64

5 SUMMARY, IMPLICATIONS, AND FUTURE RESEARCH

This work was the first to demonstrate the potential of biomimetic membranes for

desalination, has led to the discovery of pH gating in bacterial aquaporins, and has provided an

exciting first step in the determination of membrane protein structure in biomimetic membranes.

It has also led to extensions that have applications in understanding membrane protein insertion

in block copolymers, membrane protein gas transport studies, perchlorate removal, carbon

capture and. This chapter summarizes these outcomes of my dissertation research, looks at its

broad relevance, introduces some additional projects that resulted from this work, and provides

suggestions on future research.

5.1 HIGH PERMEABILITY AQPZ RICH MEMBRANE CAN MAKE IDEAL

DESALINATION MEMBRANES AND HIGH SENSITIVITY SENSORS.

This work started with the objective of inserting aquaporins into biomimetic block

copolymers to develop an alternative to current, polyamide-based, thin film composite RO

membranes. Aquaporins had been inserted into block copolymers prior to this study but their

activity was not tested (Stoenescu et al. 2004). I have demonstrated that the bacterial aquaporin,

Aquaporin Z, can be functionally inserted into an ABA-type, PMOXA-PDMS-PMOXA

membrane. Upon insertion of AqpZ, the permeability of this membrane increased by

approximately three orders of magnitude (protein to polymer molar ratio of 1:50, Figure 5.1).

Additionally, these membranes were shown to reject salt, urea, glycerol and glucose.

Flat membranes are most common for desalination and sensor applications. However,

most work with PMOXA-PDMS-PMOXA membranes with inserted membrane proteins has

been performed on the vesicle configuration (Nardin et al. 2000; Graff et al. 2002; Ho et al.

2004; Stoenescu et al. 2004; Kumar et al. 2007; Onaca et al. 2008; Graff et al. 2009;

Grzelakowski et al. 2009). A large part of this work was focused on developing a flat

configuration of AqpZ-rich block copolymer membranes. This is described in Chapter 4. The

highly packed polymer films achieved in these studies were small (up to 500 nm) and further

optimization would be needed to develop larger areas that would be practical for applications.

65

Figure 5.1: a) Insertion of AqpZ into block copolymer membranes increases permeability by an order of magnitude. b) AqpZ based membranes have higher permeability than currently used membranes. FO is commercial forward osmosis membrane, while RO is commercial reverse osmosis desalination membrane. PB-PEO is Polybutadiene Polyethylene diblock polymer. ABA represents the polymer vesicles used in study and ABA+ AqpZ represents the polymer vesicles with incorporated AqpZ at 1:50 molar ratio (see Section 2 for details).

5.1.1 Extensions

Determining insertion efficiency of membrane proteins in block copolymers. A major

experimental unknown regarding insertion of membrane proteins in lipid or polymer membranes

is the question of how many membrane proteins are inserted per unit membrane area. Efforts are

currently underway in our research group to covalently label individual aquaporins with suitable

fluorophores and determine the number of aquaporins inserted per vesicle using Fluorescence

Correlation Spectroscopy techniques modified from those presented in Rigler et al (Rigler and

Meier 2006). This work will have relevance to determining the optimum strategy for inserting

membrane proteins in block polymers. More broadly, it would provide clues to understanding

the phenomenon of membrane-membrane protein hydrophobic mismatch, its role in limiting

insertion of membrane proteins, and its possible effect on the activity of inserted membrane

proteins (Andersen and Koeppe 2007; Graff et al. 2009).

66

5.1.2 Implications

This work is proof of principle for using a biomimetic approach to develop vastly

improved desalination membranes. I have shown that incorporating AqpZ molecules into block

copolymers can increase the permeability of these polymers by orders or magnitude. More

importantly, this enhanced permeability is approximately 2-3 orders of magnitude higher than

commercial desalination membranes (Figure 5.1 (b)) while maintaining similar or higher salt

rejection capabilities. Additionally, the polymer used for this part of the study was highly

impermeable when compared to other block copolymers such as the PB-PEO membrane (Figure

5.1 (b)). This ensures that after protein insertion, flow is mostly through aquaporins and not the

membrane matrix, thus decreasing the possibility of solute leakage through the membrane

matrix. All of the above point to the promise of developing block copolymers with inserted

Aquaporins as desalination membranes.

High density membrane protein arrays may have practical applications in sensing. I

have demonstrated that protein packed structures including vesicles and small flat sheets can be

assembled. Such high density arrays have relevance to applications where a high concentration

of membrane proteins would be beneficial. In sensor applications based on membrane proteins

(Cornell et al. 1997; Martin 2007; Nielsen 2009) increased sensitivity could result from the

increased signal to noise ratio that would result from “multiplexing” multiple membrane

proteins. Similarly, in transport applications, a high density of membrane proteins would result

in enhanced transport (Kumar et al. 2007; Nielsen 2009).

5.1.3 Future Work

I have shown proof-of-concept for use of block copolymer membranes for desalination.

Further, by making templated, protein-rich films; I have been able to show that a large amount of

aquaporins can be packed into polymer films. This makes such a system attractive in terms of

the permeabilities that can be expected. However, scale up of these systems and development of

appropriate support systems for molecularly thin membranes will be a major challenge. Some

steps were made in this direction using a templating approach described in Chapter 4. However,

other efforts, including those where immobilized or deposited vesicles could be used to form

membranes, should also be pursued as these may have the ability to cover larger areas (albeit at a

lower AqpZ density). Several filtration experiments were conducted with AqpZ rich vesicles

67

deposited on an alumina filter. No rejection of salts was observed. This may be due to the lack

of sealing between the vesicles and filter pores. Thus methods where proper sealing can be

achieved should be developed.

Another question that needs to be addressed is the stability of the protein-polymer system

developed in this work. It has been reported that the chemical and mechanical stability of block

co-polymers are superior to lipid bilayer membranes. This claim is supported by studies on

bending modulus where block copolymer vesicles were aspirated into pipettes and the

deformation measured (Discher et al. 1999). Other methods should be used to further quantify

the mechanical properties and stability of block copolymer membranes. The stability of block

copolymers can be greatly enhanced by incorporating crosslinkable groups such as methacrylate

and performing crosslinking reactions. In this work (reported in Appendix C) I have tested a

method to determine the rate and extent of UV crosslinking of methacrylated PMOXA-PDMS-

PMOXA polymers using Fourier Transform Infrared (FTIR) Spectroscopy. However, these

experiments were conducted on solid polymer films. Vesicle stability tests should be conducted

before and after crosslinking. An approach to determining mechanical stability of such vesicles

in an aqueous suspension would be to evaluate change in size and/or polydispersivity (indicating

rupture) by light scattering after subjecting the vesicle suspension to varying shear rates in a cone

on plate viscometer. Additionally, osmotic shock of different magnitudes can be used to

determine the “burst pressure” of these vesicles. This experiment however needs a careful

control on the rate of mixing (such as that achieved in a stopped flow apparatus) as both the

magnitude and rate of application of osmotic pressure is relevant to bursting of vesicles. Also

such experiments could be done on vesicles stored under different conditions and over time to

determine stability.

More broadly, this work could be extended to incorporate membrane proteins with specific

transport and sensing mechanisms. Specific applications that I have begun exploring are CO2

capture (described in section 5.2.3) and perchlorate transport and degradation (described in

section 5.4.1). In terms of sensing the most promising membrane protein candidates would be

the ones currently being engineered by the Bayley group for sensing various analytes such as

organic molecules, divalent ions (Gu et al. 1999; Braha et al. 2000; Bayley and Cremer 2001)

and even DNA sequencing (Howorka et al. 2001).

68

5.2 TRIBLOCK COPOLYMERS CAN BE USED AS A PLATFORM FOR STUDYING

MEMBRANE PROTEIN PROPERTIES.

In this work ABA block copolymers were used to study the response of AqpZ to pH.

Permeability of AqpZ-ABA membranes decreased with pH from the neutral range to an acidic

range (Figure 7.2 (a)). This permeability decrease was found to be reversible with subsequent

increase in pH indicating an AqpZ gating mechanism. This gating was demonstrated in E coli

cells, and it is physiologically relevant based on experiments with wild type and mutant E. coli

strains. E. coli cells overexpressing aqpZ exhibited reduced survival under both acid and

simultaneous acid and hypoosmotic shock.

Figure 5.2. (a) pH gating of AqpZ observed in block copolymer. It was seen that the permeability of block copolymer vesicles decrease reversibly between pH 6 and 4. (b) AqpZ – ABA vesicles are stable over a wide pH range as seen from light scattering results on vesicles stored overnight at the indicated pH values (x-axis).

5.2.1 Extensions

Study of gas transport in membrane proteins using block co-polymers. Membrane

protein-mediated water and ion transport is studied by incorporation into lipid bilayers.

However, gas permeabilities of lipids are high, making it difficult to conclusively show gas

transport of incorporated proteins (Hill et al. 2004). In particular, CO2 transport across

aquaporins has been a controversial topic (Verkman 2002). The high gas permeability of lipid

membranes should preclude the need for specialized membrane proteins. Additionally, the use

of AQPs reconstituted in lipids for permeability assays has further contributed to this controversy

69

because the observed differences in permeability between lipids alone and lipids with aquaporins

is small . We are studying CO2 transport in block copolymers with incorporated aquaporins. In

preliminary studies, the CO2 permeabilities of these membranes were orders of magnitude lower

than lipid bilayers (10-6 cm/s vs. 10-3 cm/s) making this a sensitive system. In future studies

tailoring of membrane permeabilities using specific membrane proteins could be investigated.

5.2.2 Implications

Triblock copolymers can provide a stable platform for studying membrane protein

properties. PMOXA-PDMS-PMOXA polymer was used to study the permeability of AqpZ at

pH values from 2 to 12. The vesicles formed at these different pH values were stable (Figure 5.2

(b)) and even survived high pressure (~8 bar) stopped flow experiments followed by recovery

using centrifugation assisted membrane concentration. These results are a definite improvement

over lipid vesicles. In particular, lipids have high permeability for water and gases, making it

difficult to study transport of these molecules (Hill et al. 2004; Wang et al. 2007). Lipid vesicles

can also be strongly affected by pH, ionic strength, and temperature (Grit and Crommelin 1993)

complicating studies that need to be conducted under these conditions. The use of biomimetic

block copolymers to investigate a broader range of membrane protein properties provides an

excellent alternative to the lipid-based system. Further, the use of stable polymers makes it

feasible for such hybrid protein polymer systems to be used in engineering applications.

pH gating of microbial AqpZ is physiologically relevant and may be important for cell

survival in mammalian digestive tracts. Many microorganisms contain aquaporins, and some

microbial aquaporins transport water rapidly and specifically (Calamita et al. 1995; Bonhivers et

al. 1998; Hernandez-Castro et al. 2003; Shapiguzov et al. 2005), just like their eukaryotic

counterparts. However, because microorganisms have large surface area to volume ratios,

allowing fast diffusive transport, the physiological relevance of channel-mediated water transport

in microorganisms is unknown (Hill et al. 2004; Tanghe et al. 2006). Genetic analysis has not

resolved the issue, as elimination of microbial aquaporins typically results in subtle defects

(Calamita et al. 1998; Soupene et al. 2002; Hernandez-Castro et al. 2003; Shapiguzov et al.

2005), and in some cases even improves growth (Bonhivers et al. 1998). Experiments with

AqpZ mutants showed that AqpZ gating may have a role in bacterial survival under conditions of

hypoosmotic and acid shock. Figure 5.3 presents a proposed model for the role of Aquaporins

70

in preventing cell death when exposed to combined acid and osmotic shock. The acid and

combined acid and osmotic shock conditions considered here for whole cells are similar to

conditions E. coli and other pathogens might be exposed to upon passage through a host

digestive system; therefore, insights into bacterial survival under these conditions have relevance

to public health and food safety (Diez-Gonzalez et al. 1998).

Figure 5.3: Model for coordinated activity of aquaporins and mechanosensitive channels under pH and osmotic downshift conditions. Upon hypoosmotic shock aquaporins mediate rapid water transport into the cell, leading to rapid swelling. To prevent cells from bursting, mechanosensitive channels open and allow small solutes to be released. Under acid shock, mechanosensitive channel activation is lowered. The cell is therefore especially vulnerable to combined acid and hypoosmotic shock, unless water permeability of AqpZ is also reduced under acidic conditions.

5.2.3 Future Work

This work has shown that using block copolymers to incorporate membrane properties

allows flexibility in investigating a broader range of properties for these important biomolecules.

Using pH-stable block copolymers led to the discovery of pH gating in AqpZ in block copolymer

membranes and confirmation of its existence in E. coli cells. pH gating has been reported in

71

eukaryotic aquaporins, however the residues involved in gating in these aquaporins are not

conserved in AqpZ (Figure 5.4). Further work on determining the molecular mechanism of

AqpZ should be conducted as it is not clear from its structure and homology with other gated

aquaporins what residues could be involved in this behavior. Single point mutations of

protonated residues with pKa’s in the physiological range, including Histidine residues –His 56,

His 61, His 124, His 150, and His 174 should be conducted and the resulting strains should be

subjected to permeability measurements under different pH conditions to determine if pH gating

can be abolished by change in one or more residues. This will provide an indication of which

residues could be involved in the observed pH gating. Additionally, these mutated proteins

could be purified and reconstituted into block copolymer vesicles to provide further

demonstration of gating and the residues involved in gating.

Similar work could use this proposed system to study relevant properties of other

membrane proteins. An interesting case would be to study mechanosensitive channels in block

copolymers. Since the distribution of membrane stresses in a block copolymer would be

different than in a lipid membrane due to the difference in hydrophobic mismatch magnitudes, it

would be interesting to correlate the relationship between osmotic pressure required for pore

activation with the level of hydrophobic mismatch. This will provide more insight into the

gating of mechanosensitive channels and the mechanisms of osmoprotection provided by these

important membrane proteins.

Another research area would be to find candidate membrane proteins where the catalytic

activity of the protein could be enhanced from stresses induced by hydrophobic mismatch. An

excellent recent example is the NADH Ubiquinone Oxoreductase protein that was found to have

increased activity (~two times) with increasing hydrophobic block thickness (from 20 to 120

units of PDMS) (Graff et al. 2009).

5.3 BLOCK COPOLYMERS CAN BE USED FOR DETERMINING PROTEIN

STRUCTURE.

In addition to providing a platform for incorporation of membrane protein, these polymers

may provide a method for determining protein structure. In this work, a PB-PEO diblock

copolymer with a nickel functionalized hydrophilic block (PEO) (Nehring et al. 2009) was used

to provide a stable interfacial film for immobilizing AqpZ or mixed micelles of AqpZ, detergent

72

and ABA polymer. Immobilized AqpZ could be directly imaged on these films using electron

microscopy (Figure 5.4(a)). Additionally, when detergent was slowly removed, flat AqpZ

packed layers of ABA polymer were formed. These structures diffract electrons and seem to

indicate a tetragonal crystal structure (Figure 5.4(b)). A tetragonal crystal structure may be

indicative of AqpZ crystals as all reported aquaporin 2D crystals have a tetragonal unit cell

(Ringler et al. 1999; Braun et al. 2000; Gonen et al. 2004; Kukulski et al. 2005; Schenk et al.

2005; Viadiu 2007; Viadiu 2007; Ho et al. 2009). 2D crystal formation of AqpZ in block

copolymers, if confirmed, would be a major breakthrough as described in 5.3.1.

Figure 5.4: (a) Templating of AqpZ tetramers on Ni NTA block copolymer film. Inset shows a schematic of the templating system. (b) 2 D crystals of AqpZ formed in PMOXA PDMS triblock copolymer using templating block copolymers. Inset shows the electron diffraction pattern indicating a tetragonal crystal structure.

5.3.1 Implications

In this study membrane proteins could be immobilized on the surface of a chelating

diblock polymer monolayer or within the matrix of a triblock copolymer. Both of these findings

have implications for protein structure determination as described in the paragraphs below.

Membrane proteins selectively immobilized on the surface of a stable polymer

monolayer can be used to determine structure using single-particle cryo-electron microscopy.

73

The Ni-NTA polymer could be used to successfully immobilize AqpZ molecules (Figure 5.4

(b)). This has excellent application for preparing samples for imaging using single particle,

three-dimensional, cryogenic electron microscopy (cryo-EM). In single particle cryo-EM,

samples deposited on a Transmission Electron Microscopy (TEM) grid are plunge-frozen in

liquid ethane to form a thin layer of vitreous ice thus preserving the native hydrated structure of

biological macromolecules. Images of single macromoelcules can then be taken under cryogenic

conditions and averaged to obtain 3D structures of molecules. To this effect, Kelly et al have

developed the concept of an “affinity grid” where Ni –chelating lipids are deposited onto a TEM

grid, dried and stored (Kelly et al. 2008; Kelly et al. 2008a). This grid can then be directly

placed on a small sample of a crude cell lysate with added imidazole to directly recover Histidine

tagged proteins for direct imaging using cryo-EM. The Ni NTA polymer used in this work

forms a stable templating monolayer and is an excellent candidate to be used similarly. The

higher molecular weight of this polymer indicates a higher detergent resistance and overall

higher mechanical and chemical stability. For the “affinity grid” loss of the lipid monolayer to

glycerol and/or detergents limits some of the functionality of this method (Kelly et al. 2008a).

Preliminary work with these grids using polymer monolayers seems to indicate high stability

when used for membrane protein templating.

2D crystallization of membrane protein arrays in block copolymers may be a stable

platform for membrane protein structure determination. 2D crystallization of proteins in block

copolymers is an important finding of this work. 2D protein-crystals, after careful imaging using

low dose cryo-electron microscopy, can be used to determine the 3D structure of membrane

proteins in synthetic membranes (Henderson and Unwin 1975; Hankamer et al. 2007). In single

particle 3D cryo-EM each particle needs to be large enough and present in different orientations

to allow reconstruction. Conversely, 2D crystals present perfectly aligned particles in a grid,

making the mathematical process of structure reconstruction easier and possible for smaller

membrane proteins. Thus, the ability to form 2D crystals opens the door to structure

determination of membrane proteins in these polymers. The structure of AqpZ and many other

membrane proteins is known in lipids. Lipids represent the native state of membrane proteins

and structures obtained are considered the native structures of these important proteins. In this

work and future work, membrane proteins inserted in block copolymers is envisaged as a

platform for developing technologies for environmental and other applications. Further studies

74

need to be conducted to determine the structure of various candidate membrane proteins in these

materials in order to determine the suitability and properties for specific membrane-protein based

devices. Since membrane protein structure directly dictates function a careful structural

comparison of membrane proteins in lipids and the block polymer system could allow us to

explain differences in activity, or more importantly design increases or variations in activity of

these proteins (Andersen and Koeppe 2007; Graff et al. 2009). Moreover, systems could be

designed to provide conformational strain on incorporated proteins to obtain desired function or

level of activity.

5.3.2 Future Work

I have obtained promising results for crystallizing membrane proteins in triblock

copolymers obtained using the templating approach. Electron diffraction of these samples

indicates that there is crystallinity in the samples that may be a result of AqpZ crystallization,

particularly because the diffraction pattern points towards a p4 crystal such as that expected

from an Aquaporin 2D crystal (Ringler et al. 1999). However, as discussed in Chapter 4, while

promising, the images and diffraction patterns are a bit unusual. Further work should include

confirming that the crystals formed are indeed protein-polymer 2D crystals, or just pure polymer

2D crystals. If final structure of the protein in block copolymers is to be determined, additional

efforts should be directed towards making larger crystals, using other methods including dialysis.

Alternative methods of transfer to the TEM grid and better imaging techniques including the use

of cryogenic and low-dose electron microscopy techniques under various tilt conditions should

be utilized to obtain information that could lead to structure determination.

Accurate structure determination of proteins in polymers can provide a molecular explanation for

anomalous effects seen after incorporation of membrane proteins into block copolymers and

provide a basis for design of novel functional materials. Thus, attempts should be made to from

2D crystals of such proteins where a large change in conformation is suspected from activity

measurements, and that are of interest for applications.

Validation of the potential of the affinity grid technique utilizing stable Ni-NTA polymers would

to contribute to fundamental science of single particle 3D cryo-EM. In this regard, the

templating polymer should be further optimized with proteins of known structure and amenable

to 3D cryo-EM.

75

5.4 MEMBRANE VESICLES CAN BE USED FOR CONDUCTING REACTIONS ON

SURFACES, ENVIRONMENTAL REMEDIATION AND CARBON CAPTURE.

Immobilized vesicles can be used for creating surfaces with novel properties that can be

used to engineer systems for applications. In order to illustrate this concept, I participated in

collaborative work on vesicles permeabilized using a membrane protein (OmpF) and containing

the enzyme acid phosphatase. Further, the block copolymer vesicle membrane contained biotin,

and use of biotin-avidin interactions allowed controlled patterning of these vesicles on a surface

(summarized in Figure 5.5). This system can be considered an analogue of biofilm reactors

commonly used in environmental applications, where cells immobilized on surfaces are used for

degrading contaminants. Use of a fluorescent substrate enabled measurement of reaction rates by

monitoring fluorescence change. This work has been published (Grzelakowski et al. 2009) and

demonstrates how a biofilm-reactor mimic can be designed using block copolymer vesicles with

active enzymes.

Proteo-vesicle

Streptavidin

Biotin

Acid phosphatase

Elf 97® substrateBSA

Figure 5.5: Immobilized nanoreactors. Polymer nanoreactors are permeabilized with the channel OmpF and encapsulate the enzyme acid phosphatase. The enzyme substrate (ELF 97) enters the nanoreactor through OmpF and is converted by the acid phosphatase to a fluorescent product which can be monitored to determine reaction rate. The hydrophilic block of the triblock copolymer forming the nanoreactor is functionalized with biotin. A glass surface is functionalized with bovine serum albumin – biotin which binds streptavidin which then forms the link between this surface and the vesicle surface immobilizing it.

76

5.4.1 Extensions

Nanoreactors for environmental remediation of perchlorate. In this work, we have

encapsulated perchlorate degrading enzymes from specialized bacteria into block copolymer

vesicles. We also incorporated a purified outer membrane channel protein (OmpF) to allow

perchlorate to enter these vesicles (Figure 5.6). Work is currently underway to demonstrate that

perchlorate enters these vesicles through OmpF channels and is degraded. Future work includes

purification and insertion of a specific, active perchlorate transporter – the Sodium Iodide

Symporter (NIS). NIS transports perchlorate against its concentration gradient (uphill transport)

by coupling its transport to a downhill sodium gradient. Thus, the use of this transporter will

allow a high level of control of concentration of perchlorate within these nanoreactors based on

the sodium gradient utilized. The proposed system may have several advantages over currently

used biological treatment approaches as discussed in the following subsection. Further, the

ability to immobilize these nanoreactors to create a perchlorate degrading surface would be a

direct application for environmental engineering.

Figure 5.6: Perchlorate reducing nanoreactors

5.4.2 Implications

Environmental remediation using functionalized, immobilized vesicles could be an

excellent alternative to traditional biological treatment. These hybrid biological/synthetic

systems could be used for contaminants for which biological treatment is the only sustainable

option and where maintaining suitable reactor conditions is challenging. Additionally, the

77

system described above provides the flexibility of using genetically engineered proteins without

the risk of accidental release and propagation of genetically modified DNA in the environment.

Use of a CO2 impermeable block-copolymer membrane for inserting membrane

proteins could help in developing technologies for carbon capture. Our ongoing work with

block copolymer gas permeability suggests that these materials might have extremely low

permeabilities for gas. Incorporating an active transporter for CO2 can thus lead to concentration

of CO2 inside a block copolymer based vesicle. Enzymes could then convert this CO2 into

organic compounds and lead to CO2 capture. This idea is developed further in the next

subsection.

The approach proposed in this subsection could be applied in for any compound where a

specific transporter exists. Additionally, the block copolymer vesicle system allows us to create

cells with a combination of functions that do not exist in nature. This is illustrated by the use of

NIS (a mammalian protein) to concentrate perchlorate while using bacterial enzymes to degrade

it in a single synthetic cell.

5.4.3 Future Work

The feasibility of perchlorate reduction is being studied using nanoreactors with

encapsulated cell derived enzymes. Further work should include the purification of NIS, its

insertion into these block copolymers and activity studies. The insertion of membrane proteins

in the ABA block polymer system could be in either direction. Unlike the bidirectional AqpZ

and OmpF proteins, when directional active proteins (such as NIS) are inserted into such a

system, there will be difficulty in maintaining a stable concentration within these nanoreactors as

there will be constant influx as well as efflux of perchlorate over small time periods depending

on the concentration gradients set up. However, asymmetric ABC-type polymers can be used for

directional insertion and would be valuable in such cases. For the perchlorate system, the

enzymes used also need to be in a reduced state. Thus approaches to provide an external electron

donor should be investigated.

The proposed approach should be extended to other micropollutants or mixtures of

pollutants in order to understand the broad applicability of this technology. Additionally, on the

fundamental side, the effect of confinement of enzymes in a small volume should be further

studied to determine the effect of molecular crowding on enzyme activity. Further, use of

78

reactive surfaces made with such nanoreactors should be further investigated with environmental

applications; scale-up of such systems should be studied.

Ongoing work on gas transport in aquaporins incorporated into block copolymer could be

extended to develop a system for CO2 capture and conversion. This work would mimic the CO2

concentrating mechanisms (CCM) in plant cells and cyanobacteria. The central feature of this

CCM is the organelle carboxysome which is enclosed in a highly impermeable membrane.

Concentration within organelles is achieved with the use of active carbon dioxide transporters

such as the Na+/HCO3- transporter (Romero et al. 1997). This could be mimicked by inserting

the Na+/HCO3- transporter into block copolymer vesicle membranes to create a carboxysome

mimic. Incorporating the enzyme Rubisco within these vesicles could then convert CO2 into

long chain carbohydrates similar to the functioning of a carboxysome. This proposed scheme is

summarized in Figure 5.7.

Rubisco

HCO3‐

CO2

RubP

PGA+

Rubisco

HCO3‐

CO2

RubP

PGA+

Figure 5.7. Carboxysome- mimic vesicles. Carbonic anhydrase (CA) first converts dissolved CO2 into bicarbonate which could be transported into the centre of the organelle using active bicarbonate transporter channel proteins. The CO2 accumulated in this impermeable “artificial organelle” can then be combined with short chain sugars such as Ribulose Phosphate (RubP) and converted to long chain sugars such as phophphoglycolate (PGA) by the encapsulated enzyme Rubisco.

79

5.5 CONCLUSION

This work has demonstrated the promise of using hybrid membrane protein-polymer

systems for environmental engineering applications. In particular, the applicability to synthetic

desalination membranes is most promising and relevant. The basic approach used here may be

applied to any separation for which a specific transport protein is available or could be

engineered. It has also contributed to understanding the properties of aquaporins, in particular

AqpZ and its possible role in microbial physiology. Recent successes in immobilizing protein

molecules and in synthesizing 2D crystals of membrane proteins may provide an excellent path

to answer fundamental questions regarding the structure and function of these membrane


80

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94

APPENDIX A

Calculation of productivity index for BWRO and MF/UF membranes

95

MF/UF Index

The data for this index was obtained from Warren Johnson, US Filter Corporation, and is

shown in Table A.1. The raw data shown in columns 1 and 2 include the year and the cost per

unit production rate of membranes sold (price per kilo liters per day) by US Filter, respectively.

For the year 1999, 2001 and 2002 a similar value was calculated for the contracted value of

membranes at the Carmicheal WaterDistrict, CA

(http://www.carmichaelwd.org/watertreatmentplant.html), Otay Water Treatment Plant (not

built), Chula Vista, CA; and Columbia Heights Filtration Plant, Minnesota, MN, respectively.

The index was obtained by simply indexing the costs to the first year this information was

available (1989).

Table A-1: MF/UF Cost Index Data

Year cost/production rate Time UF/MF Index

$/KL/day years

1989 2000 0 1

1990 1500 1 0.75

1991 900 2 0.45

1992 700 3 0.35

1993 280 4 0.14

1994 270 5 0.135

1995 150 6 0.075

1996 120 7 0.06

1997 80 8 0.04

1998 50 9 0.025

1999 65 10 0.0325

2001 60 12 0.03

2002 65 13 0.0325

BWRO index

The data for this index was obtained from David Furukawa, Separation Consultants Inc, Poway,

CA and is shown in Table A.2. The raw data shown in columns 1, 2 and 4 include the year, the

cost per eight inch brackish water RO (BWRO) module, and the productivity (flux per unit

applied pressure), respectively. This module cost is indexed to the year 1980 (column 3). Using

96

http://www.carmichaelwd.org/watertreatmentplant.html

this indexed membrane module cost and diving it by productivity of the module provides a

cost/productivity number. This number is then indexed again to 1980 to obtain the BWRO

Improvement index shown in Figure 1.2 (b).

Table A-2: Brackish Water RO Cost Index Data

Year OEM cost

Indexed

membrane

Cost

Membrane

Productivity Years Cost/Productivity

Improvement

index

$/module indexed cost gfd/psi Indexed cost/productivity

1980 800 1.0 0.06 0 16.07 1

1985 650 0.8 0.07 5 11.42 0.711

1990 550 0.7 0.08 10 8.59 0.535

1995 450 0.6 0.10 15 5.75 0.358

2000 350 0.4 0.13 20 3.40 0.212

2010 300 0.375 0.17 30 2.14 0.133

97

APPENDIX B

Calculations for addition of NTA-polymer to 4 mm wells to obtain 100 Å2/molecule

98

Surface area for spreading of the NTA polymer in 4 mm diameter wells

= = 1.257 x10-5 m2

(4 / 4 3 210 )

The calculation of excess amount of polymer added is based on a lipid based Ni chelating

layer from Levy et al ( Journal of Structural Biology, 127, 44-52 (1999).

They target a surface area per molecule of 60 Angstrom2.

Calculating the number of molecules of Ni2+ chelating lipids (Ni2+-NTA-DOGS) needed

=1.257x10-5 m2/ (60x10-20) m2 = 2.094 x 1013 molecules

Or, 3.477 x 10-11 moles of chelating lipids needed to form one layer.

Now they suggest adding more than necessary for one layer but less than two layers to

make up for loss to solution due to presence of detergent in subphase

They actually add 0.5 microL of 0.1 mg/ml lipids (MW 1057.02) which in terms of moles

is

3110.1 / 0.5 10

4.73 101000 / 1057.02 /

mg ml mlmoles

mg g g mol

Thus the excess amount they added was = 4.73/3.48 = approx 1.4 times the theoretical

amount

For our case, The NTA polymer has a MW of 4754 g/mol and the target Mean Molecular

Area from Rainer’s data seems to be 100 Angstrom2.

Number of molecules of Ni2+ chelating polymer needed for one layer:

99

=1.257x10-5 m2/ (100x10-20) m2 = 1.257 x 1013 molecules

Or, 2.086 x 10-11 moles of chelating lipids needed to form one layer.

x 1.4 excess = 2.92 x10-11 moles

To add this much polymer on the surface in a volume of 0.5 microL,

we will need 0.5 microL of x mg/ml NTA polymer

3110.5 10

2.92 104754000 /

0.28

mg mlx moles

ml mg mol

mgx

ml

100

APPENDIX C

Crosslinking of methacrylated PMOXA PDMS triblock co-polymers using FTIR and the

effect of crosslinking on chemical and mechanical stability

101

Crosslinking of methacrylated PMOXA PDMS triblock co-polymers using FTIR and the

effect of crosslinking on chemical and mechanical stability

Summary: A series of experiments were conducted to systematically investigate UV-induced

crosslinking behavior of ABA type methacrylated PMOXA-PDMS triblock copolymer. A

knowledge of the kinetics and dose requirements of this process is important for application of

polymerized triblock copolymer to situations where the mechanical and chemical stability of the

polymer resulting from crosslinking would be important. Two polymers were used,

methacrylated ABA3 (PMOXA15-PDMS110-PMOXA15) and methacrylated ABA 4 (PMOXA12-

PDMS55-PMOXA12). These polymers were dissolved in chloroform and coated onto a glass

slide to make a thick film, from which chloroform was either evaporated at room temperature

(ABA3) or by using a refrigerated rotary vacuum evaporator (ABA4). Attenuated Total

Reflection – Fourier Transform Infra Red (ATR-FTIR) spectroscopy was performed on this

polymer to obtain a spectrum for the pure polymer. The polymer was then subjected to UV

irradiation from a solar simulator with UV intensity of 17mW/cm2 in the 340 - 390 nm range and

an overall intensity of 407 W/cm2 over different lengths of time. The polymer sample was

analyzed after each exposure with FTIR to determine the kinetics of crosslinking between the

methacrylated groups. Chemical stability of the crosslinked polymer was evaluated by

conducting dissolution tests in 100% ethanol. Mechanical stability tests have not been

performed, but could be done by subjecting vesicles to controlled shear conditions to determine

the maximum shear that such vesicles can withstand. It is known that lipid vesicles can

withstand a shear rate of only 500 s-1 in water, equivalent to an average shear stress of 5.01 x10-1

N/m2. It is anticipated that the even the unpolymerized vesicles will be able to withstand much

larger shear stresses than lipid vesicles and this will be further enhanced (maybe by a large

amount) by crosslinking of these vesicles by UV irradiation.

For both the polymers the FTIR spectrum shows distinct peaks for the PDMS and the PMOXA

parts of the polymer, indicating that this method is an excellent method to characterize this class

of polymers. The methacrylated ABA3 polymer seems to show a strong FTIR signal, and the

amount of crosslinking could be followed using a distinct peak. Also, after crosslinking the

polymer was extremely insoluble in 100% ethanol. Exposure to ambient light at room

102

temperature also caused crosslinking and led to insolubililty of the polymer in ethanol. The

crosslinking of ABA4 polymer, on the other hand, is difficult to analyze using FTIR. There are

no distinct peaks that evolve as a result of UV curing. This polymer also had no resistance to

100% ethanol. The level of methacrylation for this polymer seems to be low; increasing

methacrylation might lead to better UV curing and solvent resistance properties.

Materials and Methods

Sample prep

A 30-50 mg sample of polymer was dissolved in 5 ml CHCl3 and coated on a glass slide. In the

case of ABA3 it was left overnight to dry and form a thick film. For ABA 4 the glass slide was

put in a vacuum evaporator to dry off the CHCl3.

Fourier Transfer Infrared (FTIR) spectroscopy.

Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectrometry was used to

assess changes in characteristic functional groups of the methacrylated triblock co-polymer

during UV crosslinking. Analyses were performed with a NEXUS 670 FTIR spectrometer

(Thermo Nicolet Corporation, Madison, WI) equipped with a smart golden gate accessory,

DTGS-KBr detector, KBr beam-splitter and diamond crystal. An IR source at 45◦ incident angle

was employed. Samples of polymer were put in direct contact with the diamond crystal and 32-

64 scans were conducted in the range of 600 to 4000 cm-1 for each sample. The information

from these scans was averaged by the instrument to provide IR spectra which were then

analyzed.

UV exposure

The samples were exposed to UV light in a collimated beam apparatus to induce crosslinking of

the methacrylated polymers. The samples were exposed for different lengths of time (0.5 min to

3.5 hours) to provide different doses of UV irradiation for crosslinking. After each exposure the

samples were analyzed using FTIR to monitor changes in functional groups. The UV lamp was

a solar simulator with an overall intensity of 407 mW/cm2 and an intensity of 17 mW/cm2 in the

324 - 400 nm wavelength range expected to cause curing of polymers.

Data Analysis Methodology (Selection of appropriate peaks)

103

To investigate polymer crosslinking kinetics, FTIR spectra were examined to determine if

crosslinking of methacrylate groups causes an appreciable change in any part of the spectrum.

For the ABA3 polymer, UV exposure causes a large change in the intensity of the peak at 750

cm-1, so this was used to determine the kinetics of crosslinking. Usually ~810 cm-1 is used to

monitor crosslinking in acrylates (-CH our of plane bending) but this was shifted to 750 cm-1 in

our case. This is likely to be due to the large PDMA, PMOXA groups that the acrylates are

attached to. The change in area under the 750 cm-1 peak provided values for the change in

crosslinking with time.

For the ABA 4 polymer it was much more difficult to find an independent peak that would

confirm methacrylate crosslinking. No peak was seen at 750 cm-1 for this polymer. However,

the ~790 cm-1 peak increases as a result of UV exposure. We propose using this peak for

monitoring increase in crosslinking. It represents the formation of methylene bonds (-CH2)

bonds, which is usually observed at 814 cm-1. The use of this peak for the ABA polymer,

however, is not ideal for monitoring crosslinking as we do not have a way of determining

independent increase in crosslinking; but in the absence of independent peaks, it is a good

choice??.

Results and Discussion

The following presents a detailed description of the FTIR spectra of the two methacrylated

PMOXA-PDMS ABA polymers. It also describes the UV crosslinking of these two polymers by

monitoring change in the FTIR spectrum at different UV exposure time. Additionally the

chemical stability tests conducted with 100% ethanol are described, and a set of experiments are

proposed to characterize the mechanical (shear) stability of these polymers.

FTIR Spectrum of uncrosslinked polymers.

Figure C-1 presents the FTIR spectrum of the two methcrylated PDMS-PMOXA polymers. The

peaks corresponding to pure PDMS are evident in the spectrum. These peaks include the 798

cm-1 representing the Si-C stretching and CH3 rocking vibrations. The twin peaks at 1018 and

1092 cm-1 represent the Si-O-Si stretching vibrations. The peak at 1260 cm-1 represents the CH3

symmetric deformation of Si-CH3 bonds. The peak at 1632 cm-1 may represent the amide C=O

104

bond from the PMOXA. These peaks are common to ABA 3 and ABA 4 polymers. Several

differences are also seen between the two polymers. In particular the ABA3 polymer shows

several extra peaks including those at 696 cm-1, 756 cm-1, 1453 cm-1, 1493 cm-1 as well as at

1608 cm-1.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

600 800 1000 1200 1400 1600 1800 2000

wavelength (cm-1)

Ab

sorb

ance

(ar

b u

nit

s)

ABA3

ABA41

2

3 4

5

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

600 800 1000 1200 1400 1600 1800 2000

wavelength (cm-1)

Ab

sorb

ance

(ar

b u

nit

s)

ABA3

ABA41

2

3 4

5

Figure C-1: FTIR spectrum of ABA 3 and ABA 4 polymers. Peaks represent the PDMS and

PMOXA components of the block copolymer. These are identified as 1. (~798 cm-1) Si-C

stretching and CH3 rocking (from PDMS). 2. & 3. (~1018 and 1092 cm-1) Si-O-Si stretching

vibrations (from PDMS). 4. (~1260cm-1) CH3 symmetric deformation of Si-CH3 (from PDMS).

5. (~1632 cm-1) amide C=O bond (from PMOXA)

Crosslinking of ABA3

The exposure of the ABA 3 polymer to UV radiation caused a large decrease in the peak around

750 cm-1. This may represent the crosslinking of the methacrylate groups by representing a

decrease in the C-H bond out of plane bending. This peak is usually seen at ~814 cm-1 but the

presence of the large PDMS group attached to the methcrylate group may have resulted in a

shifting of this peak. The spectra of ABA3 polymer after exposure to different UV doses is

shown in Figure C-2(a).

105

The degree of crosslinking was calculated by determining the area under this peak, while its

evolution over time represents the kinetic of crosslinking for this polymer. This is shown in

Figure C-2(b).

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

600 800 1000 1200 1400 1600 1800 2000

Wavelength (cm-1

)

No

rmal

ized

Ab

sorb

ance

0 min

12.5 min

72.5 min

192.5 min

C-H out of plane bond (shifted)

0%

5%

10%

15%

20%

25%

30%

35%

0 50 100 150 200 250

UV Dose 324 -400 nm (J/cm 2)

cro

sslin

kin

g (

%)

(b)

(a)

Figure C-2: (a) FTIR Spectra of ABA 3 polymer exposed to increasing UV dose (time) (b)

Kinetics of crosslinking of ABA 3 polymer

Crosslinking of ABA 4

As seen in Figure C-3, it was much more difficult to find an independent peak that would

confirm the assumption of methacrylate crosslinking in the ABA 4 polymer. No peak was seen

106

at 750 cm-1 for this polymer. However, the ~790 cm-1 peak increased as a result of UV exposure.

We propose using this peak for monitoring increase in crosslinking. It represents the formation

of methylene bonds (-CH2-) bonds, which is usually observed at 814 cm-1.

The kinetics of crosslinking seem to show a lower level of crosslinking for this polymer as seen

from Figure C-3(b).

Figure C-3: (a) FTIR Spectra of ABA 4 polymer exposed to increasing UV dose (time) (b)

Kinetics of crosslinking of ABA 4 polymer

Chemical Stability

(b)

0%

1%

2%

3%

4%

5%

6%

7%

8%

9%

10%

0 50 100 150 200 250 300UV Dose324-400 nm (J/cm2)

cro

ss

link

ing

(%

)

0

0.1

0.2

0.3

0.4

0.5

0.6

600 700 800 900 1000 1100 1200

Wavelength (1/cm)

No

rma

lize

d A

bs

orb

anc

e

0 min

2.5 min

32.5 min

122.5 min

302.5 min

(a)

(b)

107

The ABA3 polymer completely dissolved in 100% ethanol without crosslinking. However, after

192.5 minutes of exposure to UV, less than 10% of mass was lost from 9 mg of the polymer after

two days of exposure to 100 % ethanol. Both the untreated and treated ABA 4 polymer dissolved

easily in 100% ethanol. This is consistent with the low level of crosslinking observed in ABA 4

polymer. The level of methacrylation for this polymer may have been quite low, resulting in

lower possibility for crosslinking.

Mechanical Stability: Proposed experiments.

We will subject both crosslinked and uncrosslinked polymer vesicles to increasing shear stresses

in a cone on plate viscometer and determine the highest shear rate that these vesicles can

withstand before rupture. A light scattering approach will be used to determine the point where a

large amount of the vesicles are ruptured. Observed rupture stresses can then be compared to

those reported in literature for Phosphatidyl choline (PC) lipid vesicles (~ 500 – 1000 mPa) (Liu

et al. 2000).

References

Liu, D. Z., W. Y. Chen, et al. (2000). Microcalorimetric and shear studies on the effects of

cholesterol on the physical stability of lipid vesicles. Colloids and Surfaces A: Physicochemical

and Engineering Aspects. 172: 57-67.

108

AUTHOR’S BIOGRAPHY

See attached Cirriculum Vitae

109

MANISH KUMAR 205 N. Mathews Ave, 4125

Urbana, IL 61801 (619) 917‐7392

[email protected]

EDUCATIONAL HISTORY Doctor of Philosophy in Environmental Engineering, Department of Civil and Environmental Engineering, University of Illinois, Urbana, IL, 08/2006 – 05/2010 (expected). Advisors: Drs. Mark M. Clark and Julie Zilles; Project: Biomimetic High Efficiency Desalination Membranes Master of Science in Environmental Engineering, Department of Civil and Environmental Engineering University of Illinois, Urbana, IL, 08/1998 – 01/2000. Advisor: Dr. Mark M. Clark; Project: Fouling of Ultrafiltration Membranes by Natural Organic Matter (NOM) Bachelor of Technology in Chemical Engineering, Department of Chemical Engineering National Institute of Technology, Trichy, India, 08/1994 – 05/ 1998 EXPERIENCE

EPA Science to Achieve Results (STAR) Fellow, University of Illinois, Urbana, IL, 2008 – current, Project: Development of highly‐efficient Aquaporin‐based water treatment membranes for desalination and contaminant removal Visiting Researcher, Wolfgang Meier Group and Andreas Engel Group. Department of Chemistry, University of Basel, Switzerland, Summer 2007 and Fall 2008

University of Illinois Fellow and Research Assistant, University of Illinois, Urbana, IL, 2007 ‐ 2008,

Project: Characterization of US Seawaters and Development of Standardized Protocols for Evaluation of Foulants in Seawater Reverse Osmosis Desalination (project in addition to dissertation research).

Senior Engineer, Applied Research Department, MWH, Pasadena, CA, 2001 – 2006, Expertise and Focus: Membrane water and wastewater treatment

Staff Engineer, NCS, Phoenix, AZ, 2000 – 2001, Expertise and Focus: Arsenic treatment, Membrane water and wastewater treatment

Research Assistant, University of Illinois, Urbana, IL, 1998 – 2000 Research Intern, Bhabha Atomic Research Center, Mumbai, India, Summer 1997

Research Intern, Central Electrochemical Research Institute, Karaikudi, India. 1996 – 1997

LICENSES Professional Engineer – Civil, California, P.E. License #69423, expiration July 2010

110

mailto:[email protected]

AWARDS

Richard and Mary Engelbrecht Fellowship, Department of Civil and Environmental Engineering, UIUC, 2009

Mavis Memorial Fund Scholarship, College of Engineering, UIUC, 2009

European Union Marie Curie Training Course Fellowship to attend 7th NCCR Practical Course on 2D Membrane Protein Crystallization and Observation , October 2008, Switzerland

US Environmental Protection Agency STAR Fellowship, 2008 – 2011, USEPA

Abel Wolman Fellowship, 2008‐2009, American Water Works Association

Henry Ford II Scholar, 2008, College of Engineering, UIUC

National Water Research Institute Fellowship for Advanced Water and Wastewater Treatment Technologies, 2007 – 2008, NWRI

Included in the “Incomplete list of Instructors ranked as excellent by their students, Fall 2006, UIUC” Courses CEE 347 and CEE 442 and in the "List of Instructors ranked as excellent by their students, Spring 2008, UIUC" CEE 437

Dissertation Travel Grant, Summer 2007, UIUC

University of Illinois Fellowship, 2006 – 2007, UIUC

MWH President’s Award for most Innovative Project, 2006

Oil and Natural Gas Corporation (Government of India) Scholarship, 1994 – 1998 INVENTION DISCLOSURES AND PATENT APPLICATIONS

1. Kumar, M; Meier, W.; Zilles, J.; Clark, M.; Grzelakowski, M.; Nehring, R. Highly permeable polymer

membranes, PCT patent application, filed Dec 4th, 2008, Assignee: University of Illinois, Corresponding Inventor: Manish Kumar

2. Kumar, M; Poust, S; Meier, W; Clark, M; Zilles, J; Cropek, D; McAllister, I. Environmental remediation with functionalized vesicles, disclosed to the University of Illinois, Dec 14th, 2009, Corresponding Inventor: Manish Kumar

PUBLICATIONS

1. Kumar, M; Marincel, M; Poust, S; Zilles, J. 2010. Aquaporin rich polymer membranes. In Biomimetic

membranes, Ed. Claus H. Nielsen, Springer (Invited Contribution, in preparation) 2. Kumar, M; Grzelakowski, M; Clark, M; Meier, W; Zilles, J. 2010. Biological role for bacterial

aquaporin gating discovered in block copolymer membranes (submitted) 3. Ladner, DA; Subramani, A; Kumar, M; Adham, S; and Clark, MM. 2009. Bench‐scale evaluation of

seawater desalination by reverse osmosis (in press) Desalination. 4. Grzelakowski, M., Onaca, O., Rigler, P., Kumar, M., & Meier, W. 2009 Immobilized Protein‐Polymer

Nanoreactors. Small, published online August 13, 2009. 5. Badruzzman M; Oppenheimer, J; Adham, S; and Kumar, M. 2009. Innovative Beneficial Reuses of

Reverse Osmosis Concentrate Using Bipolar Membrane Electrodialysis and Electrochlorination Processes, Journal of Membrane Science. 326 (2009) 392 – 399

6. Kumar, M; Grzelakowski, M; Zilles, J; Clark, M; Meier, W. 2007. Highly permeable polymeric membranes based on the incorporation of the functional water channel protein Aquaporin Z, Proceedings of the National Academy of Sciences. 2007;104(52):20719 – 20724. (featured on cover and in a commentary article in PNAS issue, also on Sciencedaily.com and several other sites)

111

7. Kumar, M; Adham, S; Pearce, W. 2007. Comparative seawater reverse osmosis pretreatment evaluation using bench and pilot scale testing, Journal of the American Water Works Association. 99:9: 168 – 178

8. Kumar, M; Adham, S; and DeCarolis, J. 2007. Reverse Osmosis Integrity Monitoring, Desalination, 214: 138 – 149

9. Kumar, M; Badruzzaman, M; Adham, S; Oppenheimer, J. 2007. Beneficial phosphate recovery from reverse osmosis (RO) concentrate of an integrated membrane system using polymeric ligand exchanger (PLE). Water Research 41, (10), 2211 – 2219

10. Kumar, M; Adham, S; & Pearce, W. 2007. An Integrated Approach to Pretreatment Evaluation for Seawater Reverse Osmosis using Bench and Pilot Scale Testing, in Membrane Treatment for Drinking Water and reuse Applications: A Compendium of Peer‐Reviewed Papers, Ed. Kerry Howe, AWWA, Denver Co.

11. Kumar, M; Adham, S, and Pearce, W. 2006. Development of a protocol for evaluation of new generation RO membranes, Journal of the American Water Works Association, 98, 4, 22 – 132

12. Kumar, M; Adham, S, and Pearce, W. 2006. Investigation of Seawater Reverse Osmosis Fouling And Its Relationship To Pretreatment Type, Environmental Science and Technology, 40, 2037 – 2044. (featured in a news story in ES&T)

13. Kumar, M; Adham, S, and Pearce, W. 2006. Evaluation of New Generation RO membranes for Brackish Groundwater Desalination, In Desalination of Seawater and Brackish Water, William Lauer (ed), Edition: 2006, Hardback, ISBN 1583213287

14. DeCarolis, J, Kumar, M, Adham, S, Pearce, W, Wasserman, L. 2005 Double Filtered for Freshness: An Integrated membrane System could expand water reuse options without excessive maintenance, Water Environment and Technology, March 2005, pg.41 – 47

15. Adham S; Kumar, M; and Pearce, W. 2005. Model Developed for Brackish and Reclaimed Water, Desalination and Water Reuse, 15(3)38 – 46

CO–AUTHORED & FUNDED RESEARCH PROJECTS*

1. Development of highly efficient aquaporin‐based membranes for aqueous separations. Mark Clark (PI) and Julie Zilles (Co‐PI). Funded by the National Science Foundation, Chemical, Bioengineering, Environmental, and Transport Systems, Chemical and Biological Separations, $400,000. Project # CBET 08‐28512, 2008 – 2011

2. Synthetic cells imbued with functional biomolecular machinery. Irene McAllister (PI), Mark Clark (Co‐PI) and Julie Zilles (Co‐PI). Funded by the U.S. Army Engineer Research and Development Center, 6.1 Basic Research , $750,000, 2008 – 2012

3. Development of highly‐efficient Aquaporin‐based water treatment membranes for desalination and contaminant removal, Manish Kumar (PI). Funded by the EPA Science to Achieve Results (STAR) Program (Fellowship), $111,000, 2008 – 2011

4. Aquaporin based membranes for water treatment, Mark Clark (PI) and Manish Kumar (Co‐PI). Funded by National Water Research Institute (Fellowship), $20,000, 2007 – 2009

5. Participated in 7 other winning proposals to Water Research Foundation (WRF). Water Reuse Foundation, and National Water Research Institute during industrial experience

* The University of Illinois does not allow graduate students to be listed as PI’s, however, I contributed substantially to idea and

collaboration development and writing the proposals listed.

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TEACHING AND MENTORSHIP ACTIVITIES

Volunteered to teach a short block of one or more Environmental Engineering courses in every semester of PhD study. Complete responsibility for the course materials and lectures (3 – 4 lecture sessions), creating, and grading homework assignment and exam question(s). Fall 2006, CEE 398 SEU: Sustainable Urban Engineering Spring 2007, Spring 2008 and Spring 2009, CEE 437: Water Quality Engineering Fall 2007 and Fall 2008, CEE 442: Physical Principles of Environmental Engineering Spring 2009, CEE 498WTS: Water Treatment Case Studies

Teaching Assistant for 3 courses in the department. I was responsible for all assignments, exams and grading as well as teaching several lectures. Fall 2006, CEE 442: Physical Principles of Environmental Engineering Fall 2006 and Fall 2007, CEE 437: Water Quality Engineering

Invited lecture in an advanced Physics course (with a Biophysics focus). Spring 2008, PHYS 498 PNM: Physics of Nanomachines

Committed and active member in recruiting and job mentoring activities for Illinois EES program

Recruited, mentored and hosted a Masters student from University of Basel, Department of Chemistry who conducted his Masters project research at Illinois

PROFESSIONAL MEMBERSHIPS, SERVICE AND ACTIVITIES

Board Member, The Spout Institute – A non profit focused on water quality education and for development of sustainable water treatment technologies for developing countries, 2009 – present

Co‐Chair, Gordon Research Conference, Graduate Research Symposium, Membranes‐Materials and Processes, 2010

Member, Association of Environmental Engineering and Science Professors, since 2008

Member, American Society for Microbiology, 2008 – present

Member, American Water Works Association, 1998 – present

Reviewer: Journal of Membrane Science, Environmental Science and Technology, Water Research, Journal AWWA, Desalination, ASCE ‐ Journal of Environmental Engineering, AQUA, Water Science and Technology

Member, Young Professionals Group, MWH, Pasadena, CA 2005 – 2006

Founding Board Member, Network of Indian Professionals, San Diego, 2001 – 2003

Vice Chair, Chemical Engineering Association, Trichy, India 1997 – 1998 INVITED PRESENTATIONS

1. “Biomimetic Membranes for Desalination Water Treatment”, invited presentation at the Swisstech

Conference, Nov 2008, Basel, Switzerland 2. “Testing of RO Concentrate Minimization and Beneficial Reuse Alternatives”, Southern California

Salinity Coalition Symposium organized by the US Bureau of Reclamation, May 2, 2006, Metropolitan Water District of Southern California, Los Angeles, CA

3. “Current Status of Worldwide MF/UF Full‐scale Plants”, MF IV symposium, National Water Research Institute, Mar 23, 2006, Orange, CA

4. “Current State of Desalination in California”, Feb 7, 2006, Southern Water, Brighton, UK 5. “Pretreatment Strategies for Seawater Desalination”, Orange County Water District, Sep 2005,

Fountain Valley, CA

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6. “Pretreatment Strategies for Seawater Reverse Osmosis”, Metropolitan Water District of Southern California, Sep 2005 Los Angeles, CA

CONFERENCE PRESENTATIONS

1. Kumar, M., Grzelakowski, M., Meier, W., Clark ,M., and Zilles, J. Bioinspired Membranes for

Environmental Applications, poster presented at Association for Environmental Engineering and Science Professors meeting, Iowa City, July 2009

2. Kumar, M., Grzelakowski, M., Clark ,M., Meier, W. and Zilles, J. pH Gating of the Escherichia coli Water Channel Protein, Aquaporin Z, poster presented at the American Society for Microbiology meeting, Philadephia, May 2009

3. Kumar, M., Clark M and Zilles, J., Biomimetic desalination membranes, poster presented at the Gordon Research Conference (Graduate Research Symposium) – Membranes and Materials, New London, New Hampshire, August 2008

4. Ladner, D. A.; Vardon, D. R.; Kumar, M., Clark, M. M. Fouling by algogenic organic matter in seawater reverse osmosis desalination. AWWA Annual Conference and Exibition, Atlanta, GA. June 2008

5. Kumar, M., Grzelakowski, M., Meier, W. Hochselektive Wasserfilter auf der Basis von Blockcopolymer Membranen, Macromolecular Colloquium, University of Freiburg, Institute for Macromolecular Chemistry, Freiburg, Germany, 2008 (presented by Dr. Meier)

6. Adham, S. , Oppenheimer, J., Kumar, M., Badruzzaman, M., Liu, L. 2007. Innovative Approaches to RO Concentrate Management : Beneficial Reuse and Concentrate Minimization, Proceedings of the International Desalting Association Conference, Gran Canaria, 2007

7. Kumar, M., Oppenheimer, J., Adham, S., and Kottenstette, R. Innovative Technologies for Beneficial Reuse of RO Concentrate, presented at the AWWA Annual Conference and Exhibition, 2006, San Antonio, Texas, 2006

8. Kumar, M.; Adham, S., Decarolis, J., Burbano, A. and Pearce, W. Pretreatment Evaluation For Seawater Reverse Osmosis Using Bench And Pilot Scale Testing, Presented at the AWWA Annual Conference, San Francisco, CA, 2005

9. Oppenheimer, J.; Chiu, K; DeCarolis, J.; Kumar, M.; Adham, S.; Snyder, S.; and Pearce, W.; Evaluating the Effect of UV Peroxide for Control of NDMA on Endocrine Disrupters, Pharmaceuticals, and Personal Care Products, presented at the AWWA Annual Conference 2006, San Antonio, Texas, 2006

10. Oppenheimer, J.; Kumar, M.; Badruzzaman, M.; Webb, L.; Kottenstette, R.; Adham, S. Using RO Concentrate as a Resource by Application of Innovative Technologies, presented at the AWWA Water Quality Technology Conference November, Denver, Colorado 2006

11. Adham, S.; Oppenheimer, J.; Kumar, M.; Webb, L.; Kottenstette, R. Innovative Approaches to RO Concentrate Management: Beneficial Reuse and Concentrate Minimization, presented at WEFTEC, Dallas, Texas, 2006

12. Kumar, M., Oppenheimer, J., Adham, S., and Kottenstette, R. Innovative Technologies for Beneficial Reuse of RO Concentrate, presented at the AWWA Annual Conference and Exhibition, 2006, San Antonio, Texas, 2006

13. Ladner, D.; Clark, M., M; Kumar, M.; and Adham, S. Bench‐Scale Studies to Characterize Organic Foulants in Seawater Reverse Osmosis Desalination, presented at the North American Membrane Society meeting, Chicago, Illinois May 12 – 17, 2006

14. Kumar, M.; Adham, S.; & Pearce, W. An Integrated Approach to Pretreatment Evaluation for Seawater Reverse Osmosis using Bench and Pilot Scale Testing, Presented at the AWWA Membrane Technology Conference, Phoenix, AZ, 2005

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15. Kumar, M.; Adham, S., Decarolis, J., Burbano, A. and Pearce, W. Pretreatment Evaluation For Seawater Reverse Osmosis Using Bench And Pilot Scale Testing, Presented at the AWWA Annual Conference, San Francisco, CA, 2005

16. Kumar, M.; Decarolis, J.; Adham, S.; Wasserman, L. Desalting of MBR treated wastewater: Integrated Membrane Systems for wastewater Reclamation, Proceedings of the California Nevada Section of AWWA, Sacramento, CA, October 2004

17. Kumar, M.; Adham, S.; & Pearce, W. Development of a Cost Model for Brackish and Reclaimed Water Desalination, Paper presented at the AWWA Annual Conference, Orlando, FL, 2004

18. Kumar, M.; DeCarolis, J.; Adham, S.; Pearce, W; Wasserman, L. Integrated Membrane Systems for Wastewater Reuse, Paper presented at the Water Environment Federation Technology (WEFTEC) conference, New Orleans, LA, 2004

19. Pearce, W.; Kumar, M. Evaluation of New Generation Low Pressure Brackish Water Reverse Osmosis Membranes, Proceedings of the California Nevada AWWA 2004 Spring Conference, April 13, 2004, Las Vegas, NV, 2004

20. Kumar, M.; Adham, S.; & Pearce, W., Development of a Spreadsheet Tool for Brackish and Reclaimed Water Membrane Desalination Costs, Paper presented at the AWWA Annual Conference, Anaheim, CA, June 2003

21. Pearce, W.; Kumar, M.; & Adham, S., Evaluation of New Generation Low Pressure Brackish Water Reverse Osmosis Membranes, Paper presented at the AWWA Annual Conference, Anaheim, CA, June 2003

22. Kumar, M.; Adham, S.; & Pearce, W., Spreadsheet Tool for Brackish and Reclaimed Water Membrane Desalination Costs, Paper presented at the AWWA Membrane Conference, Atlanta, GA, March 2003

23. Oppenheimer, J., DeCarolis, J., Kumar, M., and Gagliardo, P., Adham, S., Environmental Technology Verification of Low‐ and Medium Pressure Ultraviolet Systems, Presented at the American Water Works Association Water Quality Technology Conference. Seattle, WA, November 2002

24. Kumar, M.; Hsu, Y.; Devkota, L.; Welch, M.; and Smith, B. , Optimizing Arsenic Removal in Phoenix Area Water Treatment Plants, Paper presented at the Arizona Water Pollution Control Association (AWPCA) Annual Conference, Chandler, AZ, May 2001

25. Wang, Y.; Clark, M.M.; & Kumar, M., Characterization and fouling of Sulfonated Polyether Sulfone/Polyethersulfone – Polysulfone (SPEES/PES‐PS) UF membranes, Paper presented at the North American Membrane Society (NAMS) Conference, Denver, CO, May 2000

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