EXPANDING THE METALLOMICS TOOLBOX: DEVELOPMENT OF CHEMICAL AND BIOLOGICAL METHODS IN UNDERSTANDING COPPER BIOCHEMISTRY A Dissertation Presented to The Academic Faculty by Pritha Bagchi In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the School of Chemistry and Biochemistry Georgia Institute of Technology August 2013 Copyright © 2013 by Pritha Bagchi EXPANDING THE METALLOMICS TOOLBOX: DEVELOPMENT OF CHEMICAL AND BIOLOGICAL METHODS IN UNDERSTANDING COPPER BIOCHEMISTRY Approved by: Dr. Christoph J. Fahrni, Advisor Dr. Loren D. Williams School of Chemistry & Biochemistry School of Chemistry & Biochemistry Georgia Institute of Technology Georgia Institute of Technology Dr. Wendy L. Kelly Dr. Melissa L. Kemp School of Chemistry & Biochemistry Department of Biomedical Engineering Georgia Institute of Technology Georgia Institute of Technology Dr. Raquel Lieberman School of Chemistry & Biochemistry Georgia Institute of Technology Date Approved: April 29, 2013 To my husband and my parents ACKNOWLEDGEMENTS This journey began with a bus ride in 1998 in Kolkata, India, when I looked through the window and saw the colossal gates of the Science College of the University of Calcutta. Since then I had pursued my dream to get a doctorate degree and finally reached the moment for which I lived for the last fifteen years. I could not have dared to dream without my parents, could not have made the journey without my husband, and could not have reached the goal without my advisor. I would like to thank my advisor, Dr. Christoph Fahrni, for sparking my interest in copper biochemistry and mentoring me through my graduate studies. Dr. Fahrni has allowed me to develop this dissertation work as well as explore other areas outside of copper biology. I would also like to express my sincere gratitude towards him for believing in me through a very difficult time of my personal life. I would also like to thank my committee members: Dr. Wendy Kelly, Dr. Melissa Kemp, Dr. Raquel Lieberman, and Dr. Loren Williams for their guidance throughout these years. I would like to thank fellow graduate student Dr. M. Thomas Morgan, whose work has had a significant impact on my dissertation. Not only did he synthesize all the compounds that I have used in my work, but I strongly feel that my learning in graduate school would not have been complete without the numerous hours we spent together discussing our research. I would also like to thank Dr. Reagan McRae and Dr. Karl Huettinger, who helped me through my initial months in the Fahrni laboratory. I got invaluable support from Dr. Susan Orwig and Dr. Derrick Watkins who shared their iv experiences in protein expression and purification with me and always extended their helping hands. I cannot thank Dr. Hally Shaffer enough for her assistance; there was one time when without her help in transportation, I literally could not have done any research. I will always cherish my friendship with Dr. S. Sumalekshmy (Suma), Dr. Anna Duraj- Thatte, and Daisy Bourassa with whom I made a special bond during these years. I could not have achieved my goal without the love and support of my husband, Dr. Aniruddha Bagchi, who shared my dream as one of his own. He always walked by my side even in the darkest days of my life. I am very fortunate to get the love and blessings of my in-laws, Mr. Dipak Bagchi and Ms. Mamata Bagchi, who truly consider me as their daughter, not merely their daughter-in-law. The phrase “Thank You” cannot capture my feelings towards my parents, Mr. Chandan Maitra and Ms. Sangita Maitra, who sacrificed everything to give me the best possible education and always inspired me to aim high. I would like to take this opportunity to thank my cousins Arpita and Shuvasree and my lifelong friends Ayesha and Dipyaman for their unwavering support and unconditional friendship. v TABLE OF CONTENTS Acknowledgements .......................................................................................................... iv List of Tables .................................................................................................................... xi List of Figures .................................................................................................................. xii List of Schemes ............................................................................................................... xvi List of Abbreviations .................................................................................................... xvii Summary ...........................................................................................................................xx CHAPTER 1 INTRODUCTION ............................................................................................. 1 1.1. Interconnected Pathways in Mammalian Copper Homeostasis ........................3 1.1.1. Copper Import into the Cytosol ...................................................................5 1.1.2. Copper Chaperones ......................................................................................6 a) Metallation of Cu,Zn-SOD ........................................................................6 b) Copper Delivery to Secretory Compartments ............................................7 c) Copper Insertion into Cytochrome c Oxidase ..............................................8 1.1.3. Cellular Copper Storage .............................................................................8 1.1.4. Copper Efflux...............................................................................................9 1.2. Metal Coordination in Copper Homeostatic Proteins ......................................10 1.3. Understanding Copper Biochemistry: Bridging the Gaps ..............................13 1.4. References .............................................................................................................18 CHAPTER 2 COORDINATION CHEMISTRY OF MONOVALENT COPPER LIGANDS: A NEW SET OF COLORLESS, WATER-SOLUBLE, AND AIR-STABLE AFFINITY STANDARDS ............................................................................................................................ 26 2.1. Background ..........................................................................................................26 2.1.1. Metal Affinity of a Ligand .........................................................................27 2.1.2. Ionic Strength Dependence of Binding Affinity ........................................28 2.1.3. Concept of Apparent Stability Constant: pH-dependence of Binding Affinity .......................................................................................................30 2.1.4. Challenges in Determination of Cu(I) Stability Constants ........................33 2.2. Results and Discussion .........................................................................................41 2.2.1. Protonation Constants ................................................................................47 vi 2.2.2. Stability Constants of Copper(I) Ligands ..................................................52 2.2.2.1. Stability Constant of PEMEA ........................................................54 a) Cu(I) Affinity of PEMEA from Thermodynamic Cycle ...............57 b) Cu(I) Affinity of PEMEA from Competition with Acetonitrile ...57 2.2.2.2. Stability Constant of MCL-1 ........................................................58 a) Cu(I) Affinity of MCL-1 from Thermodynamic Cycle ................59 b) Cu(I) Affinity of MCL-1 from Competition with PEMEA ..........59 2.2.2.3. Stability Constant of BCA .............................................................62 a) Cu(I) Affinity of BCA from Competition with MCL-1 ................62 b) Cu(I) Affinity of BCA from Competition with PEMEA ..............62 2.2.2.4. Stability Constant of MCL-2 .........................................................66 2.2.2.5. Stability Constant of BCS ..............................................................67 a) Competition with DHEAMP: Cu(I) Affinity of BCS from Thermodynamic Cycle ..................................................................68 b) Cu(I) Affinity of BCS from Competition with MCL-1 ...............74 2.2.2.6. Stability Constant of MCL-3 .........................................................74 a) Cu(I) Affinity of MCL-3 from Thermodynamic Cycle ................74 b) Cu(I) Affinity of MCL-3 from Competition with BCS or BCA ....75 2.2.3. Determination of Cu(I) Affinity of a Protein using Copper Ligands ........79 2.2.3.1. Stability Constant of Cu(I) binding to CusF ..................................81 a) Cu(I) Affinity of CusF from Competition with MCL-3 ...............81 b) Cu(I) Affinity of CusF from Competition with BCA ...................82 2.3. Conclusion ............................................................................................................86 2.4. Experimental Methods ........................................................................................87 2.5. References .............................................................................................................92 CHAPTER 3 IN-SITU VISUALIZATION OF BIOLOGICAL COPPER WITH A WATER SOLUBLE FLUORESCENT PROBE: IN-GEL DETECTION OF A COPPER CHAPERONE ............................................................................................................................ 98 3.1. Background ..........................................................................................................98 3.1.1. In-Situ