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Structure and dynamics of cardiac troponin: a paramagnetic relaxation enhancement NMR study

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posted on 28.03.2022, 12:40 authored by Nicole Maree Cordina
Troponin is a large ~80 kDa, dynamic, heterotrimeric protein complex located on the thin filament of striated muscle, responsible for controlling the interaction of myosin with actin. The simple binding of Ca2+ to troponin initiates a series of protein conformational changes throughout the complex that then alter protein‐protein interactions in the muscle filament, leading to contraction. Despite the wealth of structural data on troponin, defining the molecular details of the conformational changes triggered by Ca2+ binding within the intact complex is still needed and experimentally remains a challenge. In this thesis, targeting of troponin with paramagnetic nitroxide spin labels enabled the techniques of Nuclear Magnetic Resonance (NMR) and Electron Paramagnetic Resonance (EPR) to be utilised to address fundamental questions about the structure and dynamics of the cardiac muscle troponin complex. Long‐range structural information was derived from the paramagnetic relaxation enhancement (PRE) of the NMR signal in the presence of the nitroxide spin label. Inter‐nitroxide spin distances are also obtained from continuous wave and pulsed EPR techniques. Together, both approaches provided us with a movie detailing the conformational interplay between key regions of the cardiac troponin complex in response to Ca2+ binding and phosphorylation. More importantly, incorporation of a paramagnetic species NMR have allowed us to understand the key role that dynamics plays in fine‐tuning the Ca2+ troponin switch regulatory mechanism in the cardiac isoform, and how its structure is perturbed by disease causing mutations.

History

Table of Contents

1. Introduction -- 2. The PRE effect -- 3. Materials and methods -- 4. Interdomain orientation of cardiac Troponin C characterized by paramagnetic relaxation enhancement NMR reveals a compact state -- 5. Effects of calcium binding and the hypertrophic cardiomyopathy mutation A8V on the dynamic equilibrium between closed and open conformations of the regulatory N-domain of isolated cardiac Troponin C -- 6. Tracking the calcium signal through cardiac troponin: a combined EPR and NMR study -- 7. Positioning of the cardiac specific Troponin I N-terminal extension and the effect of phosphorylation -- Appendices.

Notes

Includes bibliographical references A thesis by publication submitted in fulfillment of the requirements for the degree of Doctor of Philosophy" "August, 2013

Awarding Institution

Macquarie University

Degree Type

Thesis PhD

Degree

PhD, Macquarie University, Faculty of Science, Department of Chemistry and Biomolecular Sciences

Department, Centre or School

Department of Chemistry and Biomolecular Sciences

Year of Award

2013

Principal Supervisor

Louise Brown

Rights

Copyright Nicole Maree Cordina 2013 Copyright disclaimer: http://mq.edu.au/library/copyright

Language

English

Extent

1 online resource (xvii, 218 pages) illustrations

Former Identifiers

mq:71742 http://hdl.handle.net/1959.14/1277625