Chemical-scale manipulation of ion channels: in vivo nonsense suppression and targeted disulfide crosslinking

Thesis by: Niki Marie Zacharias

In Partial Fulfillment of the Requirements

for the Degree of

in Chemistry

California Institute of Technology

Pasadena, CA

2004

(Defended September 26, 2003) ii

” 2004

Niki Marie Zacharias

All Rights Reserved iii

Dedicated to my loving parents, George and Mary iv

Acknowledgements:

There are an enormous number of people I want to acknowledge and thank. I am very lucky to have so many great and wonderful people around me. I thank God everyday for their friendship and kindness.

First I will begin with my advisors and my committee. Dennis Dougherty and

Henry Lester have both been wonderful advisors. I have definitely enjoyed having scientific conversations with them and I know this will continue at Neurion. I especially want to thank Dennis, my main advisor, he has been a wonderful mentor and boss. He is one of the most understanding individuals I have ever met, his kindness has helped numerous graduates and undergraduates in the lab. In addition, his enthusiasm for is infectious

I have also appreciated the comments and advise I have received from my committee consisting of Dave Tirrell, Jackie Barton, and Rich Roberts. In addition, I would like to thank them for having such great graduate students as Elizabeth Boon,

Tashica Williams, Duncan Odom, Marissa Mock, Isaac Carrico, Shelly Starck, and Steve

Millward. I cannot count the number of times I have talked to people in the Tirrell,

Barton, or Roberts groups about science or life in general.

In these past six years, I have spent most of my time on the second floor of Crellin than anywhere else on Earth. Luckily, I have been able to spend that time with some wonderful lab mates. I want to thank Jennifer Ma, Wenge Zhong, Seth Miller and

Marcus Sarofim for being great mentors. I am grateful that Lintong Li started in the

Dougherty group at the same time I did. My first memories of the group are of us v struggling together to understand molecular biology and to microinject oocytes. The company of Sarah Monahan, Steve Spronk, Darren Beene, and James Petterson has been greatly appreciated. Sarah Monahan was also a great housemate and I will never forget the nights we sat around in the living room trying to make the cats jump with a laser pointer. I want thank Steve for working the night shift with me and walking me to my car at ridiculous hours of the night. Darren and James are thanked for all of their scientific advice and contributions, but they are especially thanked for their discussions on different philosophical issues like what rock bands encompass the butt-rock genre. I also want to thank Donald Elmore. He is a wonderful lab mate and friend. His English degree came in quite handy when he read my research proposals and job cover letters.

His good attitude about life is infectious. In addition, I want to thank Wendy Jen for writing her dissertation the same time I was. I believe the process of writing my dissertation was much easier because I had someone to suffer with. The tradition of having the friendliest people on campus in our group has lived on with the additions of

David Dahan, Tingwei Mu, Lori Lee, Amanda Cashin, Michael Torrice, Amy Eastwood,

Eric Rodriguez, Kiowa Bower, Julian Revie, and Joanne Xiu. I have enjoyed meeting and hanging out with all of you.

The lab mates Gabriel Brandt and Josh Maurer are in a class all by themselves.

Both of them have taught me almost everything I know about molecular biology, chemistry, and graduate school. I owe them so much, and I am very grateful for all of their help. Josh is extremely generous and very willing to help a fellow graduate whenever he can. He has been greatly missed, since his graduation. Gabe is a great fountain of knowledge – from politics, religious history, literature, geography, world vi cultures, great jazz and bluegrass musicians, to science - Gabe knows it all.

He is also a great humanitarian and a very gentle soul. I always felt privileged to be in his presence and I hope some of his knowledge and gentle nature have rubbed off on me.

I also greatly appreciated the help and friendship of members of the Lester lab like Purnima Deshpande, Cesar Labarnc, and Nivalda Rodrigues-Pinguet, Pam Fong,

John Leite, and Fraser Moss. Caltech has a knack for hiring very competent and friendly people, I especially want to thank Dian Buchness, Tom Dunn, Chris Smith, Steve Gould,

Shannon Boss, and Linda Syme for all of their assistance. Dian and Chris have been great friends and have on more than one occasion had to entertain my parents.

Many of my closest friends I have met at Caltech. My girlfriends Catherine

Baker, Heather Cox, Jennifer Johnson, Claudine Chen, Michelle Rojas, Sujata

Bhattacharyya, and Yen Bui have always been there for me. I really appreciate their companionship and I hope that I will never lose their friendship. I especially want to thank Catherine, who should be able to get a minor in chemistry just from all the manuscripts I have made her read. I also want to thank my old chemistry classmates who have become great friends - Jason Belitsky, Isaac Carrico, Elizabeth Boon, Doan Nguyen,

Soojin Kwon, and Mike Farwell. Finally, I want to thank Steve Millward who has been great companion this past year and very patient this past couple of months while I have been writing my thesis.

Another person who deserves a large thank you is Jeffery Kelly. Dr. Kelly was my undergraduate advisor at Texas A&M. It was under his advice that I applied to graduate school at Caltech. In addition, it was under his direction that I was assigned to do undergraduate research with Haimanot Beckele, who at that time was a graduate vii student in his lab. She was a wonderful role model and taught me synthetic organic chemistry. Her lessons are still remembered and appreciated.

Finally, I would like to thank my family. My parents have been a very positive influence in my life. Science is a tradition in my family, and my father made sure it stayed alive with his constant lectures on chemistry and influence on my elementary and junior high science fair projects. I will always remember him taking me to work on more than one occasion and showing me different Dow Chemical laboratories. My mother and father have always believed in me and it is through their support that my life has blossomed. I can not thank them enough. viii Abstract:

The study of the three-dimensional shape and structure-function relationships of ion channels is a very challenging field of research. Ion channels are integral-membrane proteins that when open allow ions to flux across the cell membrane. The structure and function of ion channels are dependent on the cell membrane that surrounds them.

Because an ion channel must be embedded in a cell membrane, many techniques used to probe the structure of soluble proteins cannot be used in the study of ion channels.

One versatile technique that has been shown to be quite valuable in the structure- function studies of ion channels is the in vivo nonsense suppression method for unnatural amino acid incorporation. This technique allows one to site-specifically incorporate an unnatural amino acid or hydroxy acid into a protein in a living cell. To date more than 60 amino acids and hydroxy acids have been incorporated into proteins using in vivo nonsense suppression. The method has been shown to accommodate a wide variety of unnatural amino acids and hydroxy acids. Chapter One will discusses the in vivo nonsense suppression method in greater detail.

A key component of this work is the design and synthesis of new unnatural amino acids that have novel properties. Chapter 2 discusses the synthesis and uses of 5-(o- nitrobenzyl)selenyl-2-hydroxypentanoic acid (NBSeOH). NBSeOH is used to site- specifically cleave a peptide backbone. The o-nitrobenzyl protecting group is photochemically removed to reveal a selenium anion. The selenium anion then initiates

an intramolecular SN2 displacement that cleaves the backbone of the protein. Preliminary data reveals that NBSeOH can be incorporated into a protein in vivo and in vitro, and ix photolysis of proteins and peptides containing NBSeOH does lead to protein backbone cleavage.

Chapter 4 discusses how the in vivo nonsense suppression method was used to incorporate unnatural amino acids containing a quaternary ammonium moiety to mimic the quaternary ammonium on acetylcholine. These unnatural amino acids were used to probe the nicotinic acetylcholine receptor’s binding site. These unnatural amino acids are called tethered agonists because when they were incorporated into four different positions on the nicotinic acetylcholine receptor partial opening of the channel occurred even when agonist was not present. These tethered agonists were used to obtain distance information about where acetylcholine binds within the receptor.

Another technique used to probe the structure of ion channels is targeted disulfide crosslinking. In the targeted disulfide crosslinking method, cysteine residues are introduced at various locations throughout a protein and oxidized to see whether disulfide bond formation can occur. Since only cysteine residues close in space will form a disulfide bond, this method can reveal fine structural aspects of a protein. The method was used to study the pore lining structure of the nicotinic acetylcholine receptor. Several cysteine mutants were made using mutagenesis and then studied in functional channels expressed in Xenopus oocytes. The channels were then exposed to oxidizing agents, and the ability of these mutant channels to form disulfide bonds was evaluated. Chapter 3 describes the work dealing with the targeted disulfide crosslinking experiments in the nicotinic acetylcholine receptor. x

Table of Contents:

Acknowledgements iv

Abstract viii

List of Blots xv

List of Figures xvi

List of Tables xviii

Chapter 1

Introduction

1.1 Background: 2

1.2 Bibliography: 10

Chapter 2

Synthesis and Uses of 5-(o-nitrobenzyl)selenyl-2-hydroxypentanoic acid (NBSeOH)

2.1 Introduction: 13

2.2 Synthesis: 20

2.2.1 NBSeOH and NVSeOH: 20

2.2.2 Peptide synthesis: 21 xi

2.3 Results: 24

2.3.1 Dipeptide model system: 24

2.3.2 In vitro protein incorporation – base hydrolysis: 27

2.3.3 In vitro protein incorporation – photolysis: 30

2.3.4 In vivo protein incorporation: 32

2.4 Discussion: 34

2.5 Methods: 35

2.5.1 Synthesis 35

2.5.2 Dipeptide study: 44

2.5.3 In vitro protein incorporation – base hydrolysis: 46

2.5.4 In vitro – photolysis: 47

2.5.5 In vivo suppression of NBSeOH: 48

2.7 Bibliography: 50

Chapter 3

Targeted Disulfide Crosslinking in the Nicotinic Acetylcholine Receptor

3.1 Introduction: 54

3.2 Structure of the nACh receptor: 54

3.3 Gate: 57

3.4 Targeted disulfide crosslinking: 60 xii

3.5 Electrophysiology results: 62

3.5.1 MTSEA results: 62

3.5.2 Results with copper: 63

3.5.3 Positive voltage jumps: 65

3.5.4 DTT reduction: 65

3.6 Western blot results: 67

3.6.1 Ni2+ purification and n-ethyl maleimide: 72

3.6.2 DMS control blots: 77

3.7 Radiolabeling: 78

3.8 Discussion: 81

3.8.1 Electrophysiology: 81

3.8.2 Western blots: 81

3.9 Conclusions: 82

3.10 Bibliography: 84

Chapter 4

Tethered Agonist Approach to Mapping Ion Channel Proteins – toward a structural model for the agonist binding site of the nicotinic acetylcholine receptor

4.1 Introduction: 89 xiii

4.2 In vivo nonsense suppression and the nAChR: 90

4.2.1 Tethered agonist approach: 93

4.3 Results: 98

4.3.1 Synthesis of Tyr-OnQ and Tyr-O3tBu: 98

4.3.2 Incorporation of Tyr-OnQ into the nAChR: 98

4.3.3 Incorporation of an isosteric, neutral tether: 103

4.4 Discussion: 105

4.5 An emerging model of the agonist binding site of the nAChR: 109

4.6 Conclusions: 113

4.7 Materials and methods: 113

4.7.1 Synthesis 113

4.7.2 Unnatural amino acid mutagenesis and oocytes injection: 122

4.7.3 Electrophysiology: 122

4.7.4 Bungarotoxin binding and western blot analysis: 123

4.8 Acknowledgements: 124

4.9 Bibliography: 125

Appendix

Initial Synthetic Schemes for the Synthesis of 5-(o-nitrobenzyl)selenyl-2- hydroxypentanoic acid (NBSeOH)

A.1 Introduction: 129 xiv

A.2 Methods: 134

A.3 Bibliography: 138 xv List of Blots:

Blot 2.1 29

Blot 2.2 . 29

Blot 3.1 70

Blot 3.2 . 70

Blot 3.3 70

Blot 3.4 70

Blot 3.5 71

Blot 3.6 71

Blot 3.7 71

Blot 3.8 74

Blot 3.9 74

Blot 3.10 74

Blot 3.11 74

Blot 3.12 76

Blot 3.13 76

Blot 3.14 76

Blot 3.15 76

Radiogram and Blot 3.16 80 xvi List of Figures:

Figure 1.1 3

Figure 1.2 6

Figure 1.3 8

Figure 2.1 14

Figure 2.2 15

Figure 2.3 19

Figure 2.4 19

Figure 2.5 22

Figure 2.6 25

Figure 2.7 33

Figure 3.1 56

Figure 3.2 59

Figure 4.1 91

Figure 4.2 94

Figure 4.3 97

Figure 4.4 97

Figure 4.5 102

Figure 4.6 102

Figure 4.7 104

Figure 4.8 112

Figure A.1 130 xvii

Figure A.2 131

Figure A.3 132 xviii List of Tables:

Table 2.1 25

Table 3.1 Percent of Current Inhibition after MTSEA 63

and ACh Application

Table 3.2 Percent of Current Inhibition after 63

the Application of 3 mM Cu(phen)3

Table 3.3 Percent of Current Inhibition after Application 64

of 100 mM Cu(phen)3 with ACh

Table 3.4 Percent of Current Inhibition after Application 64

of 100 mM Cu(phen)3 1

Chapter 1

Introduction 2 1.1 Background:

My graduate career has been centered on using different biochemical tools to probe protein structure-function relationships. In the case of the catalytic binding pocket of an enzyme or the ion selectivity of an ion channel, the three-dimensional structure of the protein is essential in carrying out the function of the protein. The work described here is motivated both by the desire to understand the function of nervous system proteins and the desire to develop general methods to determine the molecular basis of protein function.

Nerve impulses are propagated throughout the central nervous system and peripheral nervous system through a vast array of neurons (1). Neurons are excitable cells that can undergo rapid shifts in their electrical potential due to the flux of ions into and out of the cell. A synapse occurs where two neurons meet or a neuron interacts with peripheral organ (eye, muscle, etc.). Signal transduction at the synapse is mediated by G- protein coupled receptors and ligand-gated ion channels that recognize chemical effectors

(neuronal or hormonal) and transduce a biological action. Figure 1.1 shows a diagram of a synapse and depicts the proteins that are essential in propagating a signal from one cell to another.

To give a brief understanding of neural signal transduction, the initiation of muscle movement by the muscle-axon synapse is described (1, 2). The transmitter released by the axon terminal is acetylcholine (ACh). Muscle fibers contain nicotinic acetylcholine receptors (nAChR). The nAChR is a ligand-gated cation channel and opens when it binds its agonist (ACh). When an action potential occurs in the pre- 3

Voltage-Gated Channels Neurotransmitter Transporters

Nerve C ell

Glia G Protein-Gated Channels Postsynaptic α Cell Synapse Transmitter-Gated Channels

N

C

GTP GPCR (7-helix receptors)

Blowup of the Synapse

Figure 1.1 Molecules of neurobiology 4 synaptic motor neuron, ACh is released into the muscle-axon synapse and binds to nAChRs on the surface of the muscle fiber. Binding of ACh by the nAChR leads to a conformational change in the receptor that opens the pore of the channel, allowing positive ions to flow into the cell. When a large number of nAChRs open, the potential of the cell becomes more positive and the cell depolarizes. This depolarization opens voltage-gated Na+ channels, resulting in an influx of Na+ ions into the muscle fiber. This converts the pre-synaptic action potential into a post-synaptic action potential in the muscle fiber. Nicotinic acetylcholine receptors convert a chemical signal (ACh release) into an electrical signal by allowing ions to flow through the receptor’s pore across the cell membrane (2).

The proteins depicted in Figure 1.1 serve as ª 30% of all pharmaceutical drug targets (3). These proteins have been shown to be directly linked to psychological and senility disorders (Parkinson’s, Alzheimer’s, schizophrenia, depression) (1, 2). All of these proteins are membrane bound proteins. Their structure and function are dependent on the membrane that surrounds them. Because of this, many of the biochemical techniques used to probe the structure of soluble proteins cannot be used in the study of integral membrane proteins.

One of the most useful approaches to study ion channels and GPCRs is the combination of site-directed mutagenesis and electrophysiology, which allows for systematic structure-function studies. This capability was greatly enhanced by the adaptation of the nonsense suppression methodology for unnatural amino acid incorporation (4-6) to the heterologous expression system of Xenopus oocytes (7, 8). 5 This technique allows one to site- specifically incorporate an unnatural amino acid or hydroxy acid into a protein in a living cell. The method incorporates an unnatural amino acid or hydroxy acid into a protein by ‘tricking’ the ribosome of a cell into inserting an unnatural amino acid at a UAG (stop) codon rather than dissociating from the mRNA template and terminating translation. Combining this method with electrophysiology allows us to probe the structure-function relationships of ion channels and receptors in ways not possible with conventional mutagenesis. The method has been used to incorporate unusual amino acids bearing large side chains like a biotin analog or

‘caged’ side chains, in which, a chemical moiety on the side chain is protected by a photochemically removable group (9, 10). It has also been used to make very subtle changes to an amino acid’s side chain composition like the incorporation of tryptophan whose ring is mono-,di-, or tri-fluorinated (11, 12). The method is described in Figure

1.2.

In the method, the codon representing the amino acid where the unnatural will be incorporated into the protein of interest is mutated to an amber (TAG) stop codon. The amber stop codon is used because it is the least used of the three stop codons in Xenopus oocytes, which is the expression system used in our experiments. This construct is then transcribed in vitro to mRNA. A suppressor tRNA is then synthesized that contains the corresponding anti-codon CUA and is chemically acylated at the 3’ end with the unnatural amino acid/hydroxy acid of interest (13). Both the mRNA for the protein of interest and the suppressor tRNA are injected into Xenopus oocytes. After 24 to 48 hours electrophysiology experiments are conducted. 6

Figure 1.2 In vivo nonsense suppression technique 7 The suppressor tRNA is created using conventional molecular biology except for the last two nucleotides on the 3’ end of the tRNA. The last dinucleotides in all tRNAs are cytosine and adenosine. In the in vivo nonsense suppression technique, organic synthesis is used to produce deoxycytosine-adenosine (dCA) dinucleotide (13).

Deoxycytosine is used to reduce hydrolysis of the unnatural amino acid. The unnatural amino acid of interest is then chemically linked to dCA. This moiety is then ligated to the rest of the tRNA using RNA ligase. A full detailed procedure for the in vivo nonsense suppression technique can be found in reference (7, 8).

To date more than 60 amino acids and hydroxy acids have been incorporated into proteins using in vivo nonsense suppression (Figure 1.3) (14). A key component of this work is the design and synthesis of new unnatural amino acids that have novel properties.

Chapter 2 discusses the synthesis and uses of 5-(o-nitrobenzyl)selenyl-2- hydroxypentanoic acid (NBSeOH). NBSeOH is used to site-specifically cleave a peptide backbone. The o-nitrobenzyl protecting group is photochemically removed to reveal a

selenium anion (15, 16). The selenium anion then initiates an intramolecular SN2 displacement that cleaves the backbone ester (17).

Most of my work has been focused on the nicotinic acetylcholine receptor

(nAChR), which was discussed earlier in the chapter. Both nicotine and acetylcholine are agonists for the receptor. Chapter 4 deals with work done in collaboration with Lintong

Li, Wenge Zhong, and Caroline Gibbs to probe the acetylcholine-binding site in the nAChR (18). Lintong Li used the in vivo nonsense suppression methodology to incorporate unnatural amino acids that mimic acetylcholine into the ligand-binding 8

X HO N H H2C N N X = Br, NC, CH3, H NO2 F, NH MeO 2

CH 3 + F H3C H2 N H3C - CO2 O N X H H3C + H2 - X = OCH3, NH2, CH3 CH3 N CO2 NO 2 F, Cl, CO2H, CH3CO F O

N H3C NO2 H ACh Mimics CH F Chapter 4 3 S e S NBSeOH O N Chapter 2 F O MeO HO F CH3 F N H HO F HO N O F O F -O2C F S F N -O2C N H F N F F H N HN NH F F O O

Unnatural amino acids and hydroxy acids side chains that have been incorporated using in vivo nonsense expression

Figure 1.3 9 domain in the nAChR (Figure 1.3). Unnatural amino acids containing a quaternary ammonium that mimics the analogous functional group on acetylcholine were incorporated into the binding site. These unnatural amino acids are called tethered agonists. It was shown that when these amino acids are incorporated into four different positions on the nAChR, partial opening of the channel occurs even when agonist is not present (11, 18). The mimics were used to obtain distance information about where acetylcholine binds within the receptor.

There are many biochemical techniques that are used specifically to study membrane bound proteins. Targeted disulfide crosslinking is often used to reveal fine structural aspects of membrane bound proteins (19-21). Targeted disulfide crosslinking was used to study the pore lining structure of the nAChR. nAChRs with single cysteine mutations in each of the a subunits were expressed in Xenopus oocytes and treated with

Cu(phenanthroline)3 to catalyze the formation of a disulfide bond. Only residues that are close in space will be responsive to such treatment (21). Chapter 3 deals with all of the work done in trying to detect crosslinking between residues in the pore of the nAChR.

All three chapters deal with the uses of a particular technique to the study of a particular ion channel. However, all three techniques (NBSeOH incorporation, tethered agonists, disulfide crosslinking) could be used in theory in the study of any integral membrane bound protein. The knowledge gathered on how to implement these three techniques could be used to further understand membrane bound proteins. 10

1.2 Bibliography:

1. Hille, B. (1992) Ionic Channels of Exictable Membranes Sunderland, MA: Sinauer Associates, Inc.,

2. Kandel, E. R., and Siegelbaum, S. (1995) Transmission at the Nerve Muscle Syanpse E. R. Kandel, J. H. Schwartz, and T. M. Jessell eds. Stamford, CT: Appelton & Lange, 197-213.

3. Hopkins, A. L., and Groom, C. R. (2002) Nature Reviews: Drug Discovery 1, 727- 730.

4. Noren, C. J., Anthonycahill, S. J., Griffith, M. C., and Schultz, P. G. (1989) Science 244, 182-188.

5. Bain, J. D., Glabe, C. G., Dix, T. A., Chamberlin, A. R., and Diala, E. S. (1989) Journal of the American Chemical Society 111, 8013-8014.

6. Lodder, M., Golovine, S., and Hecht, S. M. (1997) Journal of Organic Chemistry 62, 778-779.

7. Nowak, M. W., Gallivan, J. P., Silverman, S. K., Labarca, C. G., Dougherty, D. A., and Lester, H. A. (1998) Methods in Enzymology 293, 504-529.

8. Nowak, M. W., et al. (1995) Science 268, 439-442.

9. Gallivan, J. P., Lester, H. A., and Dougherty, D. A. (1997) Chemistry & Biology 4, 739-749.

10. Anderson, R. D., Zhou, J., and Hecht, S. M. (2002) Journal of American Chemical Society 124.

11. Zhong, W., Gallivan, J. P., Zhang, Y. N., Lintong, L., Lester, H. A., and Dougherty, D. A. (1998) Proceedings of the National Academy of Sciences of the United States of America 95, 12088-12093.

12. Beene, D. L., Brandt, G. S., Zhong, W. G., Zacharias, N. M., Lester, H. A., and Dougherty, D. A. (2002) Biochemistry 41, 10262-10269.

13. Robertson, S. A., Ellman, J. A., and Schultz, P. G. (1991) Journal of American Chemical Society 113, 2722-2729. 11 14. Beene, D. L., Dougherty, D. A., and Lester, H. A. (2003) Current Opinion in Neurobiology 13, 264-270.

15. Pillai, V. N. R. (1980) Synthesis 1-26.

16. Bochet, C. G. (2002) Journal of the Chemical Society-Perkin Transactions 1 125- 142.

17. Liotta, D., Sunay, U., Santiesteban, H., and Markiewicz, W. (1981) Journal of Organic Chemistry 46, 2605-2610.

18. Li, L. T., Zhong, W. G., Zacharias, N., Gibbs, C., Lester, H. A., and Dougherty, D. A. (2001) Chemistry & Biology 8, 47-58.

19. Sun, J., and Kaback, H. R. (1997) Biochemistry 39, 11959-11965.

20. Sun, J., Kemp, C. R., and Kaback, H. R. (1998) Biochemistry 37.

21. Stoddard, B. L., Bui, J. D., and Koshland, D. E. (1992) Biochemistry 31, 11978- 11983.