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Protein Disulfide Isomerase and Prol Yl 4-Hydroxylase PROTEIN FOLDING ENZYMES: PROTEIN DISULFIDE ISOMERASE AND PROLYL 4-HYDROXYLASE by Elizabeth Alexander Kersteen A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Biochemistry) at the UNIVERSITY OF WISCONSIN-MADISON 2005 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A dissertation entitled Protein Folding Enzymes: Protein Disulfide Isomerase and Prolyl 4-Hydroxylase .~ submitted to the.Graduate School of the University of Wisconsin-Madison in partial fulfillment of the requirements for the degree of Doctor of Philosophy by Elizabeth Alexander Kersteen Date of Final Oral Examination: Marc h 24, 2005 Month & Year Degree to be awarded: December May 2005 August ************************************************************************************************** .!!l Signature, Dean of Graduate School Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. PROTEIN FOLDING ENZYMES: PROTEIN DISULFIDE ISOMERASE AND PROLYL 4-HYDROXYLASE Elizabeth Alexander Kersteen Under the supervision of Professor Ronald T. Raines At the University of Wisconsin-Madison The efficient formation of native protein structure requires an ensemble of chaperones and enzymes that improve the rate of protein conformational changes. Specific chemical modifications are catalyzed by protein folding enzymes. The native disulfide bond conformation is attained by disulfide bond formation, reduction and isomerization, processes catalyzed by protein disulfide isomerase (PDI) in the endoplasmic reticulum. Collagen, the most abundant animal protein, requires hydroxylation of proline residues for physiological stability. Prolyl4- hydroxylase (P4H) post-translationally converts specific proline residues in collagen to 4(R)- hydroxy-L-proline (Hyp). This dissertation describes advances in our understanding of the mechanisms of disulfide bond isomerization and prolyl hydroxylation. The mechanism of disulfide bond isomerization by PDI is poorly understood despite decades of research. The complexity of common substrates used to study isomerization makes it difficult to decipher details of the kinetic and chemical mechanism. Chapter Two describes the development of a novel assay for disulfide bond isomerization that uses a synthetic peptide substrate with two nonnative disulfide bonds. Isomerization is monitored directly by fluorescence resonance energy transfer. Using this new assay we have analyzed the reaction mechanism ofPDI and an active-site variant. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 11 P4H is an a2~2 tetramer in which the a subunit contains the catalytic active site and substrate binding site and the ~ subunit is PDI. Chapter Three describes the first system for the production ofP4H tetramer in Escherichia coli, which will facilitate future structural and mechanistic studies of the enzyme. Further, we describe the design of an automated assay for catalysis of prolyl hydroxylation, which will be useful for mechanistic studies and screening of inhibitors. Collagen is a triple helix composed of three strands of a repeating (Xaa-Y aa-Gly) sequence, where Xaa is often Pro and Yaa is often Hyp. Chapter Four describes the replacement of Pro and Hyp in synthetic collagen mimics with N-methyl-L-alanine, a nonnatural, acyclic tertiary amide. This substitution decreases the thermal stability of triple helices, suggesting that the conformational stability imparted by Pro and Hyp is not a result of their forming tertiary amides. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. iii Acknowledgements I am grateful for the invaluable assistance I received from many people during the preparation of this thesis. First, I would like to thank my advisor, Ron Raines, who has provided an exciting environment for scientific research. The results reported in this thesis could not have been achieved without his creativity and encouragement. His enthusiasm has helped me to overcome many hurdles along the way. Members of team PDI in the Raines laboratory have been a constant source of creativity and support. I am particularly indebted to the encouragement of Ken Woycechowsky. Ken has been a fabulous mentor and friend to me. Our many conversations about disulfide bonds and about life will be some of my fondest memories of graduate school. Seth Barrows was the inspiriation for many of the results reported in Chapter Two. Kelly Gorres has been an exciting addition to the group. Our discussions for future research directions in the lab have been fun and I look forward to hearing about her accomplishments in the upcoming years. The many members of the Raines lab, past and present, have all provided support and advise that has been important to my research. I would like to specifically thank Josh Higgin for his help with the development of the assay for hydroxylase activity described in Chapter Three and helpful discussions about the mechanism ofprolyI4-hydroxylase. Marcia Haigis was an enormous help in the early days in learning molecular biology techniques. She is also a great friend and a fun person to have in the lab. I have learned a great deal from Brian Miller regarding enzyme kinetics and mechanisms. I thank him for his encouragement and friendship. The help I received from Gary Case at the Peptide Synthesis Facility at the University of Wisconsin Biotechnology Center is much appreciated. He has been an enthusiastic source of Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. IV advice and assistance. I wish to thank Amy Harms and Grzegorz Sabat at the Mass Spectrometry Facility for their generous technical advise and services. I am grateful for the financial assistance provided by the NIH Biotechnology Training Program, the Wisconsin Alumni Research Foundation, and the Department of Biochemistry during my graduate training. The friendship and encouragement of Jake Mulligan have been invaluable to me. My time in graduate school would not have been the same without him. Jake is the most generous and genuine person I know, and I am grateful for his support. Finally, I would like to thank the unending encouragement of my family. My time in graduate school has seen many changes, but through it all our love and support of each other has been constant. I am so happy that my sister, Amy, and I have grown closer over the past few years even though we are geographically far apart. My mom's continual interest in what I've been doing is much appreciated, especially since it is so difficult to explain. My dad is at least partially responsible for nurturing my interest in science through all of those intricate grade school science projects we did together. I thank both of my parents for encouraging my individuality and creativity. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. v Table of Contents Abstract .............................................................................................................................................. i Acknowledgements .......................................................................................................................... iii Table of Contents .............................................................................................................................. v List of Tables ................................................................................................................................... vii List of Figures ................................................................................................................................ viii List of Abbreviations ........................................................................................................................ x Chapter One Introduction ....................................................................................................................................... 1 1.1 Protein Folding in the Cell ...................................................................................................... 2 1.2 Dithiol Oxidation in the Cell .................................................................................................. 8 1.3 Protein Disulfide Isomerase .................................................................................................... 9 1.4 Small Molecule Mimics ofPDI ............................................................................................ 19 1.5 Prolyl 4-Hydroxylase ............................................................................................................ 23 1.6 Collagen ................................................................................................................................ 31 1.7 A Need for More Mechanistic Understanding ..................................................................... 37 Chapter Two Catalysis of Protein Disulfide Bond Isomerization ........................................................................ 58 2.1 Abstract ................................................................................................................................. 59 2.2 Introduction ........................................................................................................................... 60 2.3 Materials and Methods .........................................................................................................
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