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Effect of phosphorylation on the structure and dynamics of cardiac troponin protein complex: a molecular view of the regulation of heart muscle under stress conditions

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posted on 28.03.2022, 15:27 authored by Ehsan Kachooei
The contraction of cardiac muscle is regulated by a large heterotrimeric complex, ~80 kDa, which is called 'Troponin' (Tn). Tn is located on the thin filament of striated muscle, playing a switch role in response to Ca2+ binding, to regulate the interaction of myosin with actin. The force of Ca2+ binding can initiate a series of conformational changes in the dynamic structure of the Tn complex which then propagate through the other components of the muscle filament, leading to contraction. Upon stress condition, and in response to β-adrenergic stimulations, Tn becomes phosphorylated in the heart which results in a faster relaxation and higher rate of contraction. Despite the wealth of structural data on troponin, the molecular details of the conformational changes triggered by Ca2+ binding and phosphorylation within the intact Tn complex is still lacking and remains an experimental challenge. In this Thesis, site directed spin labeling (SDSL), using paramagnetic nitroxide, in conjugation with Nuclear Magnetic Resonance (NMR) and Electron Paramagnetic Resonance (EPR) was used to address fundamental questions about the structure and dynamics of the cardiac troponin complex upon phosphorylation. Long‐range (10-25 Å) structural information was derived from Paramagnetic Relaxation Enhancement (PRE) effects of the NMR signal due to the presence of the nitroxide spin label. Interspin distances were also obtained from continuous wave (8 -25 Å) and pulsed EPR (DEER, up to ~70 Å) techniques. Together, both magnetic resonance approaches provided us with a structural model that details the conformational interplay between key regions of the cardiac troponin complex in response to both Ca2+ binding and phosphorylation. Knowledge of the role of phosphorylation as a secondary regulatory mechanism in fine‐tuning the cardiac Tn isoform is important for understanding why cardiac muscle is perturbed by mutations that lead to disease states such as cardiomyopathy.


Table of Contents

1. Introduction -- 2. Materials and methods -- 3. Magnetic resonance spectroscopic techniques for studying troponin structure and dynamics -- 4. Paper 1 - Phosphorylation effect on the Tni-extension region with TnC -- 5. Paper 2 - Phosphorylation effect on the interaction of the Tni switch region with TnC -- 6. Conclusion --Appendices.


Includes bibliographical references Thesis by publication.

Awarding Institution

Macquarie University

Degree Type

Thesis PhD


PhD, Macquarie University, Faculty of Science and Engineering, Department of Molecular Sciences

Department, Centre or School

Department of Molecular Sciences

Year of Award


Principal Supervisor

Louise Brown


Copyright Ehsan Kachooei 2019. Copyright disclaimer: http://mq.edu.au/library/copyright




1 online resource (xiv, 232 pages, illustrations)

Former Identifiers

mq:72128 http://hdl.handle.net/1959.14/1281656