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Hudson Udel 0060D 13 STUDIES ON OXIDATIVE PROTEIN FOLDING AND THE DEVELOPMENT OF GENETICALLY ENCODED PROBES FOR ANALYTE SPECIFIC RATIOMETRIC IMAGING by Devin A. Hudson A dissertation submitted to the Faculty of the University of Delaware in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry and Biochemistry Winter 2018 © 2018 Devin A. Hudson All Rights Reserved STUDIES ON OXIDATIVE PROTEIN FOLDING AND THE DEVELOPMENT OF GENETICALLY ENCODED PROBES FOR ANALYTE SPECIFIC RATIOMETRIC IMAGING by Devin A. Hudson Approved: __________________________________________________________ Brian J. Bahnson, Ph.D. Chair of the Department of Chemistry and Biochemistry Approved: __________________________________________________________ George H. Watson, Ph.D. Dean of the College of Arts and Sciences Approved: __________________________________________________________ Ann L. Ardis, Ph.D. Senior Vice Provost for Graduate and Professional Education I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: __________________________________________________________ Colin Thorpe, Ph.D. Professor in charge of dissertation I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: __________________________________________________________ Brian J. Bahnson, Ph.D. Member of dissertation committee I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: __________________________________________________________ John T. Koh, Ph.D. Member of dissertation committee I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: __________________________________________________________ Wilfred Chen, Ph.D. Member of dissertation committee Dedicated to the memory of my father, Michael Hudson. iv ACKNOWLEDGMENTS I would like to thank my advisor, Dr. Colin Thorpe or his guidance and support during the duration of my graduate work. Dr. Thorpe’s input and training has allowed me to grow as a scientist and researcher. I would also like to thank the current and past Thorpe lab members with whom I shared so much of my time here at the University of Delaware. I would like to thank Dr. Benjamin Israel for being my mentor and teaching me biochemistry methodology. The scientific and non-scientific discussions with my lab mates and other students in the Chemistry and Biochemistry department have made these last six years enjoyable. I would also like to thank my thesis committee members for guidance and support. I would like to acknowledge the Chemistry and Biochemistry department and the Chemistry and Biology Interface program for the opportunities that have been afforded to me. The environment that Chemistry and Biochemistry department has been built with great students, faculty, and equipment have enabled me to develop as a researcher. In addition, the collaborative environment that has fostered productive collaborations between myself and the Joseph Fox laboratory. I would also like to thank the Delaware Biotechnical Institute and Dr. Jeffery Caplan for his assistance, insight, and guidance in confocal microscopy and intercellular imaging. I would like to thank my friends, and family whom have supported me during the duration of my doctoral work. I would like to acknowledge Dr. Robert Luttrell whose advice and mentorship led me to applying for graduate school. I would especially like thank my wife, Jordan Hudson for her support and unending v encouragement that kept me going, even during some of the hardest and most trying times over the last six years. vi TABLE OF CONTENTS LIST OF TABLES ......................................................................................................... x LIST OF FIGURES ....................................................................................................... xi ABSTRACT ................................................................................................................ xiv Chapter 1 OXIDATIVE PROTEIN FOLDING .................................................................. 1 1.1 Introduction and Background .................................................................... 1 1.1.1 Protein Folding .............................................................................. 1 1.1.2 Oxidative Protein Folding ............................................................. 4 1.1.3 Oxidative folding in Prokaryotes ................................................... 4 1.1.4 Oxidative Folding in Eukaryotes ................................................... 8 1.1.5 Secretory Apparatus ...................................................................... 9 1.1.6 Mitochondrion ............................................................................. 12 1.1.7 Chloroplasts ................................................................................. 14 REFERENCES ............................................................................................................. 18 2 OXIDATIVE PROTEIN FOLDING: FROM THIOL-DISULFIDE EXCHANGE REACTIONS TO THE REDOX POISE OF THE ENDOPLASMIC RETICULUM ..................................................................... 22 2.1 Introduction ............................................................................................. 22 2.2 Chemical Aspects of Thiol-Exchange Reactions .................................... 23 2.2.1 Oxidative Protein Folding --- Enzymes and Oxidants ................ 26 2.1 PDI – Second Pathways ........................................................................... 30 2.2 PDI and Glutathione Interaction Kinetics ............................................... 32 2.3 Preamble to Redox Poise Measurements in the Mammalian ER ............ 35 2.4 ER Glutathione Redox Poise ................................................................... 36 2.5 Redox State of PDI in the Mammalian ER ............................................. 40 2.6 Chemical Trapping of PDI Redox State .................................................. 42 2.7 The Influence of PDI Redox State on In Vitro Oxidative Protein Folding ..................................................................................................... 47 vii 2.8 Future Directions ..................................................................................... 52 2.9 Envoi ........................................................................................................ 57 REFERENCES ............................................................................................................. 61 3 MIA40 IS A FACILE OXIDANT OF UNFOLDED REDUCED PROTEINS BUT SHOWS MINIMAL ISOMERASE ACTIVITY ................ 70 3.1 Introduction and Background .................................................................. 70 3.2 Experimental Procedures ......................................................................... 73 3.2.1 Materials and General Methods ................................................... 73 3.2.2 Expression and Treatment of Proteins ......................................... 73 3.2.3 RNase Activity Assay .................................................................. 74 3.2.4 Insulin Reductase Assay .............................................................. 75 3.2.5 Stopped Flow Methods ................................................................ 75 3.2.6 Sequence Analysis ....................................................................... 75 3.3 Results and Discussion ............................................................................ 76 3.3.1 Mia40/lfALR is a general catalysis of protein oxidation ............ 76 3.3.2 Reduced RNase interacts with oxidized Mia40 ........................... 80 3.3.3 Does Mia40 show significant protein disulfide isomerase activity? ....................................................................................... 82 3.4 Conclusions ............................................................................................. 87 REFERENCES ............................................................................................................. 88 4 DESIGNING FLAVOPROTEIN-GFP FUSION PROBES FOR ANALYTE-SPECIFIC RATIOMETRIC FLUORESCENCE IMAGING ...... 94 4.1 Introduction to Genetically Encode Fluorescent Protein Sensors ........... 94 4.2 Genetically Encode Fluorescent Proteins ................................................ 95 4.2.1 Limitations and Deficits in Our Current Tools to Measure Intracellular Redox Molecule Pools ............................................ 96 4.3 Materials and Methods .......................................................................... 100 4.3.1 Materials. ................................................................................... 100 4.3.2 General Procedures. ................................................................... 101 4.3.3 Protein Expression in E. coli. .................................................... 101 4.3.4 Protein Handling. ....................................................................... 102 viii 4.3.5 NADPH Reductase Activity
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