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NMR investigation into the therapeutic potential of troponin Open Access

Descriptions

Other title
Subject/Keyword
cardiomyopathy
levosimendan
troponin C
inotrope
NMR spectroscopy
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Robertson, Ian Michael
Supervisor and department
Sykes, Brian (Biochemistry)
Examining committee member and department
Solaro, R. John (Physiology and Biophysics)
Spyracopoulos, Leo (Biochemistry)
Clanachan, Alexander (Pharmacology)
MacMillan, Andrew (Biochemistry)
Department
Biochemistry
Specialization

Date accepted
2011-09-22T21:18:13Z
Graduation date
2011-11
Degree
Doctor of Philosophy
Degree level
Master's
Abstract
The pumping of the heart is controlled at the molecular level by the calcium dependent interaction between troponin C (cTnC) and troponin I (cTnI). The central role this protein-protein interaction plays in the muscle contraction cascade makes it a prime target for the development of drugs for the treatment of heart disease. In Chapters 2 and 3, we show that the natural products, EGCg and resveratrol, bind preferentially to the C-terminal domain of cTnC (cCTnC). NMR structures reveal that EGCg binds to the surface of the hydrophobic pocket of cCTnC, whereas resveratrol binds deeper in the protein, akin to the Ca2+-sensitizer, EMD 57033. The comparisons between the two structures highlight specific interactions between the compounds and cCTnC that define differences in their binding poses. The next section (Chapters 4, 5, and 6) is devoted to understanding the mechanism of drugs that target the N-terminal domain of cTnC (cNTnC). Specifically, the modulation of cTnI binding to cNTnC is entertained as the mechanism by which molecules that bind to cNTnC modulate contraction. In Chapter 4 some pharmacophores are identified and an ideal cNTnC-cTnI construct for the design of drugs is described. Chapter 5 explores the structure and function of a novel Ca2+-sensitizer, dfbp-o. We find that dfbp-o enhances cTnI binding in vitro and increases contractility in situ. This enhanced cTnI binding is postulated to originate from an electrostatic attraction between R147 of cTnI and the carboxylate moiety of dfbp-o. In Chapter 6 the synthesis and activity of some novel analogs of the inhibitor, W7, is outlined. The results support the electrostatic mechanism outlined in Chapter 5. In Chapters 7 and 8 we investigate how one can modify calcium sensitivity by changing residues on either cNTnC or cTnI. We show that the mutation L48Q stabilizes the open state of cNTnC thereby enhancing cTnI binding and contractiltity. A specific histidine on skeletal TnI has been shown to increase the calcium sensitivity of a myofilament when compared to cTnI, at low pH. In Chapter 8, we show that under acidic conditions, this histidine is protonated and its binding to cNTnC is enhanced by the appearance of an electrostatic interaction with E19 of cNTnC.
Language
English
DOI
doi:10.7939/R35M78
Rights
License granted by Ian Robertson (imr2@ualberta.ca) on 2011-09-22 (GMT): Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of the above terms. The author reserves all other publication and other rights in association with the copyright in the thesis, and except as herein provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
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