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Structural Motifs of Novel Metallothionein Proteins Western University Scholarship@Western Electronic Thesis and Dissertation Repository 4-25-2012 12:00 AM Structural Motifs of Novel Metallothionein Proteins Duncan E K Sutherland The University of Western Ontario Supervisor Dr. Martin J. Stillman The University of Western Ontario Graduate Program in Chemistry A thesis submitted in partial fulfillment of the equirr ements for the degree in Doctor of Philosophy © Duncan E K Sutherland 2012 Follow this and additional works at: https://ir.lib.uwo.ca/etd Part of the Biochemistry Commons, Inorganic Chemistry Commons, and the Molecular Biology Commons Recommended Citation Sutherland, Duncan E K, "Structural Motifs of Novel Metallothionein Proteins" (2012). Electronic Thesis and Dissertation Repository. 489. https://ir.lib.uwo.ca/etd/489 This Dissertation/Thesis is brought to you for free and open access by Scholarship@Western. It has been accepted for inclusion in Electronic Thesis and Dissertation Repository by an authorized administrator of Scholarship@Western. For more information, please contact [email protected]. i Structural Motifs of Novel Metallothionein Proteins (Spine title: Structural Motifs of Metallothionein) (Thesis format: Integrated-Article) By Duncan Ewan Keith Sutherland Graduate Program in Chemistry Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy School of Graduate and Postdoctoral Studies The University of Western Ontario London, Ontario, Canada April, 2012 © Duncan Ewan Keith Sutherland 2012 School of Graduate and Postdoctoral Studies The University of Western Ontario CERTIFICATE OF EXAMINATION Supervisor Examiners Dr. Yining Huang Dr. Martin Stillman Dr. John Honek Dr. Ken K.-C. Yeung Dr. Graeme Hunter The thesis by Duncan Ewan Keith Sutherland entitled: Structural Motifs of Novel Metallothionein Proteins is accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Date Chair of the Thesis Examination Board ii ABSTRACT Metallothioneins (MT) are a family of small cysteine rich proteins, which since their discovery in 1957, have been implicated in a range of roles including toxic metal detoxification, protection against oxidative stress, and as a metallochaperone involved in the homeostasis of both essential zinc and copper. The most well studied member of the family is the mammalian MT, which consists of two domains: a β-domain with 9 cysteine residues, which sequesters 3 Cd2+/Zn2+ or 6 Cu+ ions, and an α-domain with 11 cysteine residues, which sequesters 4 Cd2+/Zn2+ or 6 Cu+ ions. Despite over half a century of research, the exact functions of MT are still unknown but must be related to its metalation status. Several areas that are not well studied, but that could lead to the assignment of function include 1) the determination of the exact mechanism of metalation and the structural characterization of 2) submetalated and 3) supermetalated forms of MT. Together these three areas of study will provide a more comprehensive understanding of the potential metalation chemistries of MT in vivo. Towards this goal, the following thesis presents electrospray ionization mass spectrometric (ESI MS) data showing that the mechanism of metalation of MT is noncooperative. That is metalation events occur independently of each other, allowing for partially metalated species to exist in vivo. Further metalation studies using the isolated domains of MT as metal ion competitors against the full MT protein have yielded evidence that a new Zn5-MT exists in which both domains ‘coalesce.’ In addition, NMR and CD spectroscopy, coupled with ESI mass spectrometric data, have shown the existence of a new ‘supermetalated’ Cd8-MT, which also results in a ‘coalescence’ of both domains. Taken together these results indicate that 1) partially metalated forms of the protein are in fact stable and 2) the traditional structural view of MT, where both domains act in isolation, is in fact the exceptional case and that under conditions of metal ion deficiency and excess, both domains interact with each other. Keywords: Metallothionein, cadmium, zinc, mechanism of metalation, competitive metalation, metal-induced folding, supermetalated metallothionein, ESI mass spectrometry, NMR spectroscopy, CD spectroscopy iii CO-AUTHORSHIP STATEMENT The following thesis contains material from previously published manuscripts. Dr. Martin Stillman is coauthor of all the published papers and was responsible for supervising Duncan Sutherland. For all chapters in which a portion has been published, Duncan Sutherland wrote the first draft of the paper. Dr. Martin Stillman was involved in all levels of publication having major roles in both editing and revising the published manuscripts. For Chapter 2 and 3, Duncan Sutherland was solely responsible for acquiring all cadmium metalation data. Ms. Kelly Summers is gratefully acknowledged for acquiring zinc metalation data (Chapter 4), as well as her help with the preparation of related figures. Further, Duncan Sutherland was responsible for all experimental considerations and training of Ms. Kelly Summers, who acquired the ESI MS data and was a coauthor of the manuscript published. For Chapter 5 and 6, Dr. Mathew Willans (NMR facility manager, UWO) is gratefully acknowledged for his role in determining the parameters for and helping to acquire data from 113cadmium-NMR samples. For his contribution, Dr. Mathew Willans is listed as a coauthor on all associated manuscripts. Duncan Sutherland was responsible for all sample preparation, interpretation of data, figure development and writing of manuscripts. iv ACKNOWLEDGEMENTS I would like to acknowledge my parents, Ian and Katherine Sutherland, as well as my brother, Thomas Sutherland, for their support of my education (both graduate and undergraduate). Without their unwaivering encouragement, none of this would have been possible. I would also like to thank members of the Stillman bioinorganic group, both past and present for being exceptional labmates. Specifically Thanh Ngu for being my role model in graduate school, Michael Tiedemann for being a great friend and as a great resource in both undergraduate and graduate school, Kelly Summers for her collaborative work, and finally Tyler Pinter for his novel experimental approach and unique mechanical abilities. Many thanks are also given to the staff of the Electronic Shop (John Vanstone, Warren Lindsay, Barakat Misk and Jon Aukema) and the Chemstore staff (Marylou Hart, Sherrie McPhee and DonYakobchuk). I am greatly indebted to Doug Hairsine for training and advice on the ESI MS and the Department of Chemistry (and its entire staff) for my experience as a both an undergraduate and gradute student. Last but not least, I would like to thank Professor Martin Stillman for his support of my research efforts and the opportunities he gave me to collaborate with other research groups, involve myself as a volunteer for a series of international chemistry conferences (CanBIC-1, -2 and -3) and many international conference opportunities. v TABLE OF CONTENTS STRUCTURAL MOTIFS OF NOVEL METALLOTHIONEIN PROTEINS................................................. I CERTIFICATE OF EXAMINATION ............................................................................................................... II ABSTRACT ........................................................................................................................................................ III CO-AUTHORSHIP STATEMENT .................................................................................................................. IV ACKNOWLEDGEMENTS ................................................................................................................................. V LIST OF FIGURES .............................................................................................................................................. X LIST OF TABLES .............................................................................................................................................. XI LIST OF ABBREVIATIONS AND DEFINITIONS ...................................................................................... XII CHAPTER 1. INTRODUCTION ................................................................................................................. 1 1.1 METALLOTHIONEIN ................................................................................................................................ 1 1.2 FUNCTION .............................................................................................................................................. 3 1.2.1 Metal ion homeostasis ...................................................................................................................... 3 1.2.2 Toxic metal detoxification ................................................................................................................. 4 1.2.3 Protection against oxidative stress ................................................................................................... 7 1.3 STRUCTURAL CHARACTERIZATION ........................................................................................................ 8 1.4 ZINC AND CADMIUM BINDING TO MT .................................................................................................. 10 1.5 COPPER BINDING TO MT ...................................................................................................................... 11 1.6 MERCURY BINDING TO MT .................................................................................................................
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