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SMALL-MOLECULE CATALYSIS of NATIVE DISULFIDE BOND FORMATION in PROTEINS by Kenneth J. Woycechowsky a Dissenation Submitted in Pa

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SMALL-MOLECULE CATALYSIS of NATIVE DISULFIDE BOND FORMATION in PROTEINS by Kenneth J. Woycechowsky a Dissenation Submitted in Pa SMALL-MOLECULE CATALYSIS OF NATIVE DISULFIDE BOND FORMATION IN PROTEINS by Kenneth J. Woycechowsky A dissenation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Biochemistry) at the UNIVERSITY OF WISCONSIN-MADISON 2002 A dissertation entitled Small-Molecule Ca~alysis of Na~ive Disulfide Bond Formation in Proteins 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 Kenneth J. Woycechowsky Date of Final Oral Examination: 19 September 2002 Month & Year Degree to be awarded: December 2002 May August **** .... **************** .... ** ...... ************** ........... "1"II_Ir'IIII;;IISIJii:iAh;;;;;?f~D~i:s:sertation Readers: Signature, Dean of Graduate School , 1 Abstract Native disulfide bond formation is essential for the folding of many proteins. The enzyme protein disulfide isomerase (POI) catalyzes native disulfide bond formation using a Cys-Gly-His­ Cys active site. The active-site properties of POI (thiol pK. =6.7 and disulfide E;O' =-180 mY) are critical for efficient catalysis. This Dissertation describes the design, synthesis, and characterization of small-molecule catalysts that mimic the active-site properties of POI. Chapter Two describes a small-molecule dithiol that has disulfide bond isomerization activity both in vitro and in vivo. The dithiol trans-I,2-bis(mercaptoacetamido)cyclohexane (BMC) has a thiol pKa value of 8.3 and a disulfide E;O' value of -240 mV.ln vitro, BMC increases the folding efficiency of disulfide--scrambled ribonuclease A (sRNase A). Addition of BMC to the growth medium of yeast cells causes an increase in the heterologous secretion of Schizosaccharomyces pombe acid phosphatase. which contains eight disulfide bonds. Chapter Three describes a tripeptide that is a functional mimic of POI. The disulfide bond stability of the peptide Cys-Gly-Cys-NH2 (E;O' =-167 m V) matches closely that of the POI active sites. In vitro, the reduced form of this peptide has higher disulfide bond isomerization activity than does the reduced form of BMC. The CGC sequence was incorporated into a protein scaffold via deletion of the proline residue from the CGPC active site of Escherichia coli thioredoxin (Trx). This deletion destabilizes the active-site disulfide bond ofTrx and confers substantial disulfide bond isomerization activity. Chapter Four describes the synthesis of an immobilized dithiol that is useful for protein folding in vitro. Tris(2-mercaptoacetamidoethyl)amine was synthesized and coupled to two ii different solid supports: NovaSyn~ TG Br resin (TentaGel resin, a co-polymer of styrene and bromine-functionalized polyethylene glycol) and styrene-glycidyl methacrylate microspheres. TentaGel presenting dithiol shows little disulfide bond isomerization activity with a sRNase A substrate, due to sterie inaccessibility of the dithiols within the interior of the TentaGel resin. In contrast, microspheres present dithiol with a high surface density and show disulfide bond isomerization activity that is both substantial and greater than microspheres presenting a monothiol. III Acknowledgements This Thesis was not produced in isolation, and I am grateful for the assistance of many people along the way. Ronald T. Raines has been a wonderful advisor and the source of many ideas put forth in this Thesis. I appreciate his generosity and patience with me during my studies. He has provided a creative environment in which to undertake scientific research. Prof. Dane Wittrup (M.I.T.) collected the protein secretion data presented in Chapter 2. During his rotation in the Raines lab, Brad Hook helped to collect data that appears in Chapter 4. The efforts of the other Raines lab members of team POI are greatly appreciated. I am indebted to Drs. Martha Laboissiere and Peter Chivers for sharing their knowledge and expertise with me as I started this project. George Caucutt and Emily Pfieffer were talented undergraduates and the opportunity to guide their research enhanced my graduate experience. Seth Barrows was always willing to exchange points of view. Betsy Kersteen proofread parts of this Thesis and I have enjoyed sharing my ideas and frustrations with her. The other members of the Raines Lab have also helped me during my tenure here. In particular, Dr. Jonathan Hodges and Man Soellner served as good friends as well as editors of Chapters 3 and 4, respectively. Dr. Chiwook Park provided the idea for the symmetric trithiol used in Chapter 4 and also willingly answered my unending stream of chemical and biological questions. Dr. Lynn Bretscher and Brad Nilsson provided technical assistance with my syntheses. Drs. Brad Kelemen, Marcia Haigis, and Tony Klink provided a continuous source of advice and friendship. Bryan Smith solved many computer problems for me; I will miss his iv munificence and enthusiasm. Roz Sweeney made the Raines lab a more fun and well-managed place in which to work and continues to be a valued friend. I wish to acknowledge the financial support, during my graduate training, of the Wisconsin Alumni Research Foundation, the NIH Chemistry-Biology Interface Training Program, and the Department of Biochemistry. The support and encouragement of Courtney Cater have meant the world to me. Finally, I would like to thank my family. During his stay here, my brother Doug improved the quality of my life in Madison. I credit my parents for nurturing my academic pursuits, which helped me to reach this point. v Table of Contents Abstract ....................................................................................................................................... i Acknowledgements ................................................................................................................... iii Table of Contents ....................................................................................................................... v L1st· 0 fF'IgureS .......................................................................................................................... Vll.. List of Tables ............................................................................................................................. ix List of Abbreviations,. ................................................................................................................. x Chapter One Introduction ....................................... , '" ..................................................................................... 1 1.1 Disulfide Bonds and Protein Folding ................................................................................. 2 1.2 Pathway of Disulfide Bond Formation in the Endoplasmic Reticulum ............................... 3 1.3 POI Structure .................................................................................................................... 6 1.4 Catalytic Mechanism of POI ............................................................................................. 8 1.5 Role of Protein Disulfide Isomerase in the Cell ............................................................... 11 1.6 Thioredoxin as a Substitute for Protein Disulfide Isomerase ............................................ 14 1.7 Properties of the CXXC Motif......................................................................................... 15 1.8 Strategies for Improving Native Disulfide Bond Formation in Vivo ................................. 17 1.9 Strategies for Improving Native Disulfide Bond Formation in Vitro ................................ 20 Chapter Two A Small-Molecule Catalyst of Protein Folding in Vitro and in Vivo .......................................... 37 2.1 Abstract .......................................................................................................................... 38 2.2 Introduction .................................................................................................................... 39 2.3 Materials and Methods .................................................................................................... 41 vi 2.4 Results ............................................................................................................................ 46 2.5 Discussion ....................................................................................................................... 49 2.6 Prospectus ....................................................................................................................... 56 2.7 Significance .................................................................................................................... 57 Chapter Three The CXC Motif - A Functional Mimic of the Protein Disulfide Isomerase Active Site ............. 70 3.1 Abstract .......................................................................................................................... 71 3.2 Introduction .................................................................................................................... 72 3.3 Materials and Methods .................................................................................................... 74 3.4 Results ......................................., .................................................................................... 79 3.5 Discussion ....................................................................................................................... 84 3.6 Prospectus ......................................................................................................................
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