Saturday, May 31, 2025
5/31/2025
Post translational modifications tune cardiac myosin - ABSTRACT We hypothesize that the post-translational modification (PTM) of the heavy chain of human cardiac myosin head (S1) regulates myosin allosteric transition between two distinct states, active, ready to interact with actin and the sequestered super-relaxed state when myosin S1 bound to the proximal S2 domain of myosin tail and sterically constrained from the interaction with actin. Rapid and reversible switch between myosin structural states could be a regulatory mechanism of muscle activation when more active heads become available in a sarcomere for force production. The proposal is based on (a) our recent success in the expression and purification of human cardiac myosin using the C2C12 murine myoblasts expression system, and (b) on the successful application of a toolset of complementary biophysical methods to study myosin and actomyosin kinetics, myosin motility, and myosin S1-S2 interactions. We propose to study three selected PTM-mimetic mutants of the human cardiac myosin. We will examine the effect of mutations on the kinetics of the actomyosin cycle, the binding affinity of myosin S1 and proximal S2, the population of myosin in the sequestered SRX state, and unloaded and loaded in vitro motility of the PTM-mimetic myosin constructs. Two aims of the proposal are complementary. The changed kinetics of the actomyosin cycle will be detected in the transient kinetics experiments and as the changed velocity of actin filament in the unloaded in vitro motility assay. The affected stability of the SRX state will be assessed directly in the single turnover ATP assay and will be detected as changed ensemble force of myosin bed in the loaded in vitro motility assay. As the result, we will examine our hypothesis that PTM destabilizes the SRX state of myosin and will characterize the effect of PTM on human cardiac myosin kinetics and motility.
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