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View of the Pichia Expression System STRUCTURE-FUNCTION STUDY OF CELLULAR IRON CHEMISTRY DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By JIA HUANG ***** The Ohio State University 2009 Dissertation Committee: Approved by Professor James A. Cowan, Advisor Professor Karin Musier-Forsyth _________________________________ Advisor Professor Ross E. Dalbey Graduate Program in Chemistry ABSTRACT Iron-sulfur clusters are small inorganic cofactors found in all kingdoms of life. The iron-sulfur cluster biogenesis is a complex system which involves a surprisingly large group of proteins. In human cells, frataxin is believed to recruit ferrous iron from the labile iron pool and subsequently deliver it to the scaffold protein human ISU where the iron-sulfur clusters will form. Deficiency in cellular frataxin production results in a human disease, Friedreich's ataxia which affects 1 in 50,000 humans. By using mutagenesis, the activities of frataxin mutants were investigated. We found that frataxin may have a pool of potential sites that can stably bind an iron center when bridged to a variety of physiological targets; there may not be unique binding loci, but rather a number of locations that provide flexibility in the binding of physiological partner proteins. Frataxin has been found to repair the damaged cluster on mitochondrial aconitase. We herein investigated its activity on cytosolic aconitase, IRP1, which can register the iron level and is important for the iron homeostasis of human cells. Frataxin can bind to cytosolic aconitase and repair the damaged clusters ([3Fe-4S]) into [4Fe-4S] clusters. Recently, we reported a potential role for the N-terminus (residues 56-77) as a structural switch to control access to the acidic surface. We also characterized a high affinity iron-binding site in the full-length frataxin that promotes an autocatalytic ii cleavage of the N-terminal domain. Herein we reported the cloning and overexpression of human mitochondrial HscB, a J-type co-chaperone, and demonstrated an interaction between human frataxin and human HscB by cross-linking experiments and ITC. HscB was also shown to promote cleavage of the N-terminal domain of full-length human frataxin. Human ISU protein contains three highly conserved cysteine residues (C44, C70, and C113) that mutagenesis studies suggested to be directly coordinated to the cluster. The fourth ligand is still unclear. H112 was mutated into alanine or aspartate, and the effects of the mutation were evaluated. We found that H112 might be the fourth ligand that helps to stabilize the cluster, however, it was not absolutely needed for the formation of cluster; it might be the ligand that helps to deliver iron. Different lines of results from other labs suggested that ependymin might be involved in long-term memory formation and neurodegeneration. Human ependymin was cloned for expression in E coli, yeast and insect cell. The protein from overexpression using pET-32 was extracted, purified and refolded. The biochemical characteristics were evaluated. iii To my father and mother, Xinyao Huang and Zhihua Dou iv ACKNOWLEDGMENTS I thank my adviser, Dr. J. A. Cowan, for providing a stimulating and flexible environment that helps me to develop independent thinking and research skills. I thank him for providing intellectual guidance and being continuously patient and understanding. I am also thankful for his effort on assisting me with scientific writing. I would also like to thank those that have served on my dissertation committee. I would like to thank all members of Cowan research group, including Taejin Yoon, Yan Jin, Wenbin Qi, Shu-pao Wu, Yushi Liu, Chun-An Chen, Seth Bradford, Lalintip Hocharoen, Shu Ding , Jeff Joyner, Jaye Murdoch, and Monica Luo, for all the help that you offered. I would like to thank my parents. They have been a constant source of support. I am very grateful to my husband, Dr. Jin Wang, for his patience and effort of being a computer support and formatting this dissertation. I greatly appreciate the funding that I received from the Department of Chemistry. v VITA February 5, 1981 ..................................................... Born, Yichang, Hubei, China 2003......................................................................... B. S. Chemistry Wuhan University 2005 – 2009............................................................. Teaching Assistant The Ohio State University 2003 – 2009............................................................. Research Assistant The Ohio State University PUBLICATIONS Research Publications 1. Huang J, Dizin E, Cowan JA (2008) Mapping iron binding sites on human frataxin: implications for cluster assembly on the ISU Fe-S cluster scaffold protein. J. Biol Inorg Chem; 13, 825-36. 2. Huang J, Cowan JA. (2009) Iron-sulfur cluster biosynthesis: role of a semi- conserved histidine. Chem Commun. 2009, 3071-3. FIELDS OF STUDY Major Field: Chemistry vi TABLE OF CONTENTS Page Abstract ............................................................................................................................... ii Dedication .......................................................................................................................... iv Acknowledgments............................................................................................................... v Vita ..................................................................................................................................... vi List of Tables ................................................................................................................... xiv List of Figures ................................................................................................................... xv List of Abbreviations ..................................................................................................... xviii CHAPTERS: 1. Introduction ............................................................................................................. 1 1.1. Importance of Iron-Sulfur Clusters .................................................................. 1 1.2. Pathways of Iron-Sulfur Cluster Biogenesis .................................................... 3 1.3. The ISC Assembly System .............................................................................. 3 1.3.1. Role of the Scaffold Protein IscU ............................................................. 4 1.3.2. Iron Binding and Delivery Protein Frataxin ............................................. 6 1.3.3. Role of Cysteine Desulfurase .................................................................... 6 1.3.4. Role of Chaperones ................................................................................... 7 1.3.5. Other Important Components of ISC Assembly System .......................... 8 vii 1.4. Human Diseases Caused by Defects in the Iron-Sulfur Cluster Biosynthesis . 9 1.5. References for Chapter 1 ................................................................................. 9 2. Mapping Iron Binding Sites on Human Frataxin: Implications for Cluster Assembly on The ISU Fe–S Cluster Scaffold Protein .......................................... 14 2.1. Introduction .................................................................................................... 14 2.2. Materials and Methods ................................................................................... 20 2.2.1. General chemicals ................................................................................... 20 2.2.2. Cloning, Expression, and Purification of His-tagged Truncated Native Frataxin ............................................................................................................. 20 2.2.3. Cloning, Expression, and Purification of Frataxin Derivatives A, B, and C ........................................................................................................................ 22 2.2.4. Expression and Purification of Apo Human ISU and D37A ISU ........... 23 2.2.5. Mass-Spectrometric Determination of Protein Molecular Mass ............ 23 2.2.6. Quantitation of Iron Binding to Frataxin and Derivatives by ITC .......... 24 2.2.7. Quantitation of Zinc Binding to Frataxin and Derivatives by ITC ......... 25 2.2.8. Quantitation of Iron Required for Frataxin–ISU Binding....................... 25 2.2.9. Fe–S Cluster Assembly on ISU Mediated by Frataxin and Derivatives . 26 2.3. Results ............................................................................................................ 27 2.3.1. Cloning, Expression, and Mass-Spectrometric Characterization of Frataxin and Derivatives ................................................................................... 27 2.3.2. Iron Binding to Native Frataxin and Derivatives .................................... 32 2.3.3. Zinc Binding to Native Frataxin ............................................................. 36 2.3.4. Evaluation of Iron-Promoted Frataxin-ISU Complex Formation by ITC ........................................................................................................................... 38 2.3.5. [2Fe–2S] Cluster Reconstitution Mediated by Native and Derivative Frataxins ............................................................................................................ 42 2.4. Discussion .....................................................................................................
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