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Title: Kinetic characterisation of disease causing mutations in the embryonic and ß-cardiac myosin motor domain
Author: Walklate, Jonathan
ISNI:       0000 0004 6061 8910
Awarding Body: University of Kent
Current Institution: University of Kent
Date of Award: 2016
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Myosin myopathies are a growing area of research not only to understand the nature of the disease and how it can occur, but also to gain insight into how the myosin molecule works. Point mutations are a great way of examining how regions of myosin interact, however, given that there are over 800 amino acids in the motor domain alone, pinpointing key residues can be challenging. The missense mutations in the myosin molecule that lead to disease are ideal then to investigate residue changes that will have an effect on the function of the motor. The expression of recombinant skeletal myosin class II molecules has only recently become possible. Previous studies into the function of the embryonic myosin isoform have shown it to be a slow type myosin similar to the β-cardiac isoform. Here stopped-flow kinetic analysis of recombinant embryonic myosin S1 showed it has a tight ADP affinity and slow ADP release, characteristic of the β-cardiac myosin. Analysis of the three most common mutations in the embryonic myosin that cause Freeman-Sheldon syndrome (R672H, R672C, and T178I) showed a significantly reduced ATP hydrolysis, and ATPase Vmax and KM. Modelling of the cycle found that the mutations will be detached from actin for longer due to reduced ATP hydrolysis rate and a slower estimated phosphate release step. Another more common myopathy is hypertrophic cardiomyopathy (HCM) which can be cause by mutations in a multitude of sarcomeric proteins, most notably the β-cardiac myosin. HCM is usually found in adolescents and young adults; however cases are beginning to emerge involving young children. Stopped-flow kinetic analysis of one of these mutations, H251N, shows more significant effects on the myosin function than 'adult' HCM mutations, including; a weaker ADP affinity, tighter ATP affinity, and slower detachment from actin rate constant. However the difference in severity is not apparently clear from the stopped-flow data alone. These results highlight new key areas on the myosin molecule that are essential for its correct function. The myosin motor is an intricate machine with multiple parts that need further investigation to truly understand its function and the impact of disease causing mutations.
Supervisor: Geeves, Michael Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available