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Defining a Molecular Mechanism for Lead Toxicity Via Calcium-Binding

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Defining a Molecular Mechanism for Lead Toxicity Via Calcium-Binding Georgia State University ScholarWorks @ Georgia State University Chemistry Dissertations Department of Chemistry Spring 5-7-2011 Defining a Molecular Mechanism for Lead oT xicity via Calcium- Binding Proteins Michael Kirberger Follow this and additional works at: https://scholarworks.gsu.edu/chemistry_diss Part of the Chemistry Commons Recommended Citation Kirberger, Michael, "Defining a Molecular Mechanism for Lead oT xicity via Calcium-Binding Proteins." Dissertation, Georgia State University, 2011. https://scholarworks.gsu.edu/chemistry_diss/53 This Dissertation is brought to you for free and open access by the Department of Chemistry at ScholarWorks @ Georgia State University. It has been accepted for inclusion in Chemistry Dissertations by an authorized administrator of ScholarWorks @ Georgia State University. For more information, please contact [email protected]. DEFINING A MOLECULAR MECHANISM FOR LEAD TOXICITY VIA CALCIUM- BINDING PROTEINS by MICHAEL KIRBERGER Under the Direction of Professor Jenny J. Yang ABSTRACT Essential metals like Ca2+ and Zn2+ play critical roles in biological processes through protein interactions. Conversely, non-essential metals (e.g., Gd3+ and Pb2+) also interact with proteins, often with toxic effects. Molecular metal toxicity is assumed to be due to ionic displacement, and studies have demonstrated that Pb2+ replaces Zn2+, Ca2+ and other essential metals in proteins. The focus of this work was to compare protein Ca2+ and Pb2+ -binding sites and to investigate a mechanism of Pb2+ toxicity in Ca2+-binding proteins, particularly the intracellular trigger protein calmodulin (CaM) which binds four Ca2+ ions and interacts with numerous molecular targets via Ca2+-induced conformational change. A statistical analysis of PDB structural data for Pb2+ and Ca2+-binding (EF-hand and non-EF-hand) proteins revealed fewer binding ligands in Pb2+ sites (4 ± 2), than non-EF-Hand (6 ± 2) and EF-Hand (7 ± 1) Ca2+-binding sites. Pb2+ binds predominantly with sidechain Glu (38.4%), which is less prevalent in both non-EF-Hand (10.4%) and EF-Hand (26.6%) sites. Interestingly, analyses of proteins where Pb2+ replaces Ca2+ (calmodulin) or Zn2+ (5-aminolaevulinic acid dehydratase) revealed structural changes presumably unrelated to ionic displacement. These results suggested that Pb2+ adopts diverse binding geometries and that opportunistic binding outside of known Ca2+-binding sites may play a role in molecular metal toxicity. 2+ Ca -binding affinities (Kd) using phenylalanine and tyrosine fluorescence were found to be 1.15 ± 0.68 X 10-5 M and 2.04 ± 0.02 X 10-6 M for the N- and C-terminal 2+ domains, respectively. The Kd for Pb -binding in the N-terminal domain, 1.40 ± 0.30 X 10-6 M, was 8-fold higher than Ca2+. Binding of Pb2+ in the C-terminal domain produced a -7 -6 biphasic response with Kd values 7.34 ± 0.95 X 10 M and 1.93 ± 0.32 X 10 M, suggesting a single higher affinity Pb2+-binding site in the C-terminal domain with nearly equivalent affinity for the remaining sites. Competitive effects of Pb2+ added to Ca2+- loaded CaM were examined using multiple NMR techniques. Pb2+ was found to displace Ca2+ only in the N-terminal domain, however structural/dynamic changes were observed in the central helix apparently due to Pb2+-binding in secondary sites. These data supported our hypothesis that CaM structure and function is altered by opportunistic Pb2+-binding. INDEX WORDS: Calcium, Lead, EF-Hand, Calmodulin, Toxicity DEFINING A MOLECULAR MECHANISM FOR LEAD TOXICITY VIA CALCIUM- BINDING PROTEINS by MICHAEL KIRBERGER A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the College of Arts and Sciences Georgia State University 2011 Copyright by Michael Kirberger 2011 DEFINING A MOLECULAR MECHANISM FOR LEAD TOXICITY VIA CALCIUM- BINDING PROTEINS by MICHAEL KIRBERGER Committee Chair: Jenny Yang Committee: Markus Germann Aimin Liu Robert Wolhueter Electronic Version Approved: Office of Graduate Studies College of Arts and Sciences Georgia State University May 2011 Acknowledgements I would like to express my appreciation for the many people who have either contributed to the work presented in this dissertation, or have supported my efforts during the course of my time at Georgia State University. First and foremost I would like to thank my research advisor, Dr. Jenny J. Yang, for her support and guidance. I would also like to thank my dissertation committee members Dr. Markus Germann, Dr. Aimin Liu and Dr. Robert Wolheuter, as well as other instructors at Georgia State who have shared their knowledge with me and encouraged me to stay and complete my doctoral degree: Dr. Keith Pascoe; Dr. Laura Kibler-Herzog; and Dr. Giovanni Gadda. Additionally, I would like to express my appreciation for the members in Dr. Yang‘s lab with whom I have worked, including: Dr. Hing-Cheung Wong, Dr. Xue Wang, Dr. Ning Chen, Dr. Jin Zou, Dr. Hsiau-wei Lee, Dr. Yubin Zhou, Jingjuan Qiao, Jie Jieng, Shen Tang, Yanyi Chen, Shenghui Xue, Ling Wei and Adriana Castiblanco. Finally, I wish to thank my wife Barbara, and my children, Connor, Caoilinn, and Logan, for their love, support, and for patience while enduring this long process. Funding for this work was provided by NIH Grants GM070555, GM 62999-1 and a Molecular Basis of Disease Fellowship from Georgia State University. iv Table of Contents Acknowledgements ........................................................................................................ iv List of Figures .................................................................................................................. x List of Abbreviations ..................................................................................................... xiv 1. Introduction ............................................................................................................. 1 1.1 Metals and metal-binding in protein biochemistry ............................................ 1 1.2 Non-essential metals: toxicity and potential applications .................................. 3 1.3 Physiological effects of lead (Pb2+) toxicity ....................................................... 4 1.4 Mechanisms of metal toxicity and Pb2+-binding in proteins .............................. 4 1.5 Roles of calmodulin and other Ca2+-binding proteins in Pb2+-toxicity ................ 6 1.6 Statistical and structural analyses of Pb2+-binding and molecular toxicity ...... 12 1.7 The significant roles of metals in diagnostics and radiotherapy ...................... 15 1.8 Cell adhesion molecule CD2 as scaffold protein for RNT agent ..................... 24 1.9 Objectives of this dissertation ........................................................................ 25 1.10 Significance of this dissertation ...................................................................... 26 2 Materials and methods ......................................................................................... 29 2.1 Ca2+-binding protein statistics ........................................................................ 29 2.2 Pb2+-binding protein statistics ........................................................................ 34 2.3 Expression and purification of CaM ............................................................... 36 2.4 Expression and purification of isotopically-labeled CaM ................................. 39 2.5 Determination of CaM concentration .............................................................. 42 2.6 Methods for controlling free Ca2+ in buffers and protein samples ................... 42 2.7 Fluorescence studies ..................................................................................... 45 2.8 NMR studies .................................................................................................. 58 2.9 Equilibrium dialysis sample preparation ......................................................... 69 2.10 Sub-cloning of CD2.7E15 variants ................................................................. 71 v 3 Analyses of Ca2+-binding in proteins ................................................................... 74 3.1 Ca2+-binding proteins and Ca2+-binding sites ................................................. 74 3.2 EF-Hand superfamily ..................................................................................... 81 3.3 Canonical EF-Hand binding motif .................................................................. 82 3.4 Pseudo EF-Hand binding motif ...................................................................... 84 3.5 C2 domain ..................................................................................................... 86 3.6 Ca2+ and enzymes ......................................................................................... 87 3.7 Non-EF-hand binding sites ............................................................................ 90 3.8 Calcium in ion channels ................................................................................. 91 3.9 Statistical analysis of Ca2+-binding sites ........................................................ 93 3.10 Conclusions ................................................................................................. 107 4 Statistical analyses of Pb2+-binding in proteins ................................................ 110 4.1 Pb2+-binding protein statistics ...................................................................... 110 4.2 Ligand coordination by binding
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