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Structure of the Membrane Proximal Oxidoreductase Domain of Human Steap3, the Dominant Ferrireductase of the Erythroid Transfer

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Structure of the Membrane Proximal Oxidoreductase Domain of Human Steap3, the Dominant Ferrireductase of the Erythroid Transfer STRUCTURE OF THE MEMBRANE PROXIMAL OXIDOREDUCTASE DOMAIN OF HUMAN STEAP3, THE DOMINANT FERRIREDUCTASE OF THE ERYTHROID TRANSFERRIN CYCLE by Anoop Kumar Balakrishnan Sendamarai ` A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry MONTANA STATE UNIVERSITY Bozeman, Montana September 2009 ©COPYRIGHT by Anoop Kumar Balakrishnan Sendamarai 2009 All Rights Reserved ii APPROVAL of a dissertation submitted by Anoop Kumar Balakrishnan Sendamarai This dissertation has been read by each member of the dissertation committee and has been found to be satisfactory regarding content, English usage, format, citation, bibliographic style, and consistency, and is ready for submission to the Division of Graduate Education. Dr. C. Martin Lawrence Approved for the Department of Chemistry and Biochemistry Dr. David J. Singel Approved for the Division of Graduate Education Dr. Carl A. Fox iii STATEMENT OF PERMISSION TO USE In presenting this dissertation in partial fulfillment of the requirements for a doctoral degree at Montana State University, I agree that the Library shall make it available to borrowers under rules of the Library. I further agree that copying of this dissertation is allowable only for scholarly purposes, consistent with “fair use” as prescribed in the U.S. Copyright Law. Requests for extensive copying or reproduction of this dissertation should be referred to ProQuest Information and Learning, 300 North Zeeb Road, Ann Arbor, Michigan 48106, to whom I have granted “the exclusive right to reproduce and distribute my dissertation in and from microform along with the non- exclusive right to reproduce and distribute my abstract in any format in whole or in part.” Anoop Kumar Balakrishnan Sendamarai September 2009 iv ACKNOWLEDGEMENTS I would like to thank my teachers for their invaluable guidance all through the years. I also want to thank Dr. Martin Lawrence and the other members of the Lawrence research group at Montana State University for their constant support, encouragement, and co-operation. A special thanks to our collaborators Dr. Mark Fleming and Dr. Robert Ohgami for a chance to work on this challenging project. Finally, I would like to thank my family and friends for their unconditional love and support as they stood by me in challenging times. v TABLE OF CONTENTS 1. INTRODUCTION .......................................................................................................... 1 Iron in Biology .............................................................................................................. 1 Iron in Oxidative Stress ..........................................................................................3 Iron Requirement in Mammalian Systems ............................................................ 4 Anemia ................................................................................................................... 5 Hemochromatosis .................................................................................................. 8 Mechanism and Regulation of Mammalian Iron Uptake ............................................ 10 Absorption of Iron .................................................................................................10 Mammalian Heme Uptake .....................................................................................10 Inorganic Iron Uptake ...........................................................................................12 Transferrin Cycle ..........................................................................................................14 Transferrin Cycle in Erythropoiesis ......................................................................17 Iron Homeostasis ..........................................................................................................18 Molecular Control of Cellular Iron Homestasis ....................................................18 Systemic Iron Regulation ...................................................................................... 22 Steap3, the Major Ferrireductase in the Tf Cycle ........................................................25 Steap3 in the Transferrin Cycle ............................................................................28 Steap3, the Major Ferrireducatase in the Transferrin Cycle .................................29 Steap Family of Proteins .............................................................................................. 32 2. MATERIAL AND METHODS ....................................................................................37 Purification and Crystallization of Mouse Steap3 Oxidoreductase Domain ................37 Crystallization Trials .............................................................................................37 Designing and Cloning of Human Steap3 Oxidoreductase Domain ............................ 39 Construct Design ................................................................................................... 39 Cloning ..................................................................................................................40 Gateway Cloning ........................................................................................... 43 Expression and Purification of hu-Steap3 .................................................................... 45 Expression of hu‐Steap3 ....................................................................................... 45 Purification ............................................................................................................46 Crystallization and Structure Determination of hu-Steap3 .......................................... 47 Crystallization of Steap3 .......................................................................................47 Structure Determination of Apo‐Steap3 ...............................................................48 Structure Determination of NADPH‐Steap3 .........................................................52 PDB Deposition ....................................................................................................52 vi TABLE OF CONTENTS - CONTINUED 3. RESULTS .....................................................................................................................53 Crystallization of mouse-Steap3 ...................................................................................53 Structure of Oxidoreductase Domain of Human Steap3 ...............................................54 Introduction ...........................................................................................................54 Expression and Purification of human-Steap3 ......................................................55 Structure Determination of the Steap3 Oxidoreductase Domain ..........................58 Structure of the Steap3 Oxidoreductase Domain ..................................................61 Structure‐Function Relationships Within the Dimer ............................................66 NADPH Binding Site ............................................................................................67 Putative Flavin Binding Site .................................................................................70 Proximity of Substrate Binding Site to the Membrane .........................................72 4. DISCUSSION ...............................................................................................................74 5. CONCLUSIONS ...........................................................................................................81 6. STRUCTURAL CHARACTERIZATION OF A223, A STRUCTURAL PROTEIN FROM SULFOLOBUS TURRETED ICOSAHEDRAL VIRUS (STIV) ..............................................................................................................83 Introduction ..................................................................................................................83 Archaea the Third Domain of Life ........................................................................83 Sulfolobus Turreted Icosahedral Virus (STIV) .....................................................84 Structural Studies of STIV Proteins ......................................................................84 A223, a Structural Protein from STIV ..................................................................86 Method and Materials ...................................................................................................89 Cloning and Expression ........................................................................................89 Crystallization of A223 .........................................................................................90 Purification ....................................................................................................90 Crystallization ...............................................................................................91 Site-directed Mutation of A223 ............................................................................91 Molecular Replacement ........................................................................................94 N-terminally His Tagged Protein ..........................................................................94 Results and Discussion .................................................................................................95 Crystallization of A223 .........................................................................................95
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