Public defence in Biomedical sciences: Lok Priya Velayuthan

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Abstract

Myosin II is a molecular motor that primarily functions in muscle contraction where it generates force and motion through cyclic interaction with actin filaments, driven by ATP turnover. Gaining mechanistic insights into actomyosin energy transduction is essential, particularly in the context of mutations associated with hypertrophic cardiomyopathy (HCM), a genetic condition linked to altered cardiac function. The current approach for studying mutations in the myosin motor domain relies on protein expression in C2C12 cells using adenovirus-based transfection system. However, this method is constrained by slow turnaround time and labor-intensive protocols. This thesis presents a virus-free transfection method to express human β- cardiac myosin subfragment-1 (denoted as S1L) in C2C12 cells using commercially available chemical reagents – JetPrime and GenJet. The purified S1L proteins exhibited actin-activated ATPase and sliding velocities (using in vitro motility assay) comparable to those obtained using the virus-based system. Our new alternative method provides a faster and less complex approach for screening a wide range of HCM mutations. Furthermore, a highly miniaturized single-molecule ATPase assay was developed to leverage the advantage of the virus-free expression system. Using total internal reflection fluorescence microscopy (TIRF), fluorescent ATP turnover rate constants for myosin were determined for both basal and actin- activated conditions. The latter was obtained by crosslinking S1L proteins to surface immobilized actin filaments via EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) crosslinker. The observed turnover rates were consistent with those obtained from solution-based kinetic assays. By integrating the virus-free expression system with the single-molecule ATPase assay, several S1L point mutations, including R243E, R243E+E466R, R243C, and R243H were biochemically characterized alongside the classical HCM mutations R403Q and R453C. The R243 mutants are of particular interest, as this residue plays an important role in secondary Pi binding site and may influence the multistep process of inorganic phosphate release. Initial findings suggest that the R243C mutation could serve as a promising model for investigating orthophosphate release, potentially offering deeper insights into the molecular mechanisms underlying HCM pathogenesis.

Keywords: Human β-cardiac myosin, mutations, hypertrophic cardiomyopathy, C2C12, virus-free transfection, expression, single-molecule ATPase, actin- activated ATPase, crosslinking, basal ATPase, total internal reflection fluorescence microscopy, phosphate release.