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Functional Investigation into the Physiology and Pathology of Myosin Class II Isoforms

Johnson, Chloe Anne (2019) Functional Investigation into the Physiology and Pathology of Myosin Class II Isoforms. Doctor of Philosophy (PhD) thesis, University of Kent,. (KAR id:80793)

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Abstract

Myosin is a cytoskeletal motor that uses metabolic energy stored in ATP to do mechanical work. Muscle myosin in all mammals consists of a variety of isoforms, each expressed from its own gene. All of the striated muscle myosin sequences are highly conserved, but each myosin isoform confers distinct contractile characteristics to distinct muscle fibre types. How each myosin is tuned for its specific function is not well understood. We combined detailed biochemical characterisation with a novel kinetic modelling approach to probe how sarcomeric myosin isoforms adapt their mechanochemical cross-bridge cycle to perform different functions. The next question from this was how the sequences of these highly conserved isoforms give rise to these contractile differences. Focussing on the β cardiac myosin (MyHC-β), we investigated how the sequence of the protein can drive adaptation to changes in body mass by altering the rate of ADP-release and velocity of contraction. Bioinformatics analysis identified the sequence variants in MyHC-β that directly control its velocity and the biochemical validations are presented. This demonstrates how a protein can adapt over evolutionary time frames to meet different physiological requirements, which remains one of the fundamental questions in structural and molecular biology. Mutations in the same protein (MyHC-β) are a major cause of the life-threatening disease, Hypertrophic Cardiomyopathy (HCM). The specific mechanistic changes to myosin function that lead to this disease remain incompletely understood. We hypothesised that mutations that result in early onset disease would have more severe changes in function than do later onset mutations. Contrary to our hypothesis, no clear distinction was observed in the molecular behaviour of MyHC-β between early and late onset HCM mutations. One of the existing challenges of a study of this scale is the difficulty in producing recombinant myosin protein. This thesis will describe the development of an innovative expression system in insect cells which produce C. elegans body wall myosin in a non-muscle environment. The approach was validated by the biochemical characterisation of the resulting protein, which was found to be homologous to human MyHC-β, suggesting it could be used as a new model protein to study human disease. My thesis describes how sarcomeric myosins have fine-tuned their properties to give rise to different physiological functions, and how these processes are disrupted in diseased-states.

Item Type: Thesis (Doctor of Philosophy (PhD))
Thesis advisor: Geeves, Michael
Subjects: Q Science
Divisions: Divisions > Division of Natural Sciences > School of Biosciences
SWORD Depositor: System Moodle
Depositing User: System Moodle
Date Deposited: 15 Apr 2020 14:04 UTC
Last Modified: 20 May 2021 13:24 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/80793 (The current URI for this page, for reference purposes)
Johnson, Chloe Anne: https://orcid.org/0000-0003-0856-5041
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