Abstract

We have studied the kinetics of ATP induced actomyosin dissociation and ADP release in two mutants of beta isoform of the human cardiac myosin S1. The mutations were introduced in the Loop 1 (D208Q: K450L) and in the force-generating region (R694N) of the myosin head. Both mutants support myosin ATPase activity. We introduced these mutations to remove electrostatic interactions between charged residues in the specified domains of myosin S1. Our computational analysis showed that in the wild-type myosin, permanent salt bridges are present in the specified regions of the beta isoform. Those salt bridges are absent in alpha isoform. Since the kinetics of alpha isoform is an order of magnitude faster than the kinetics of beta isoform, we hypothesize that electrostatic interactions in the specified regions play a crucial role in myosin kinetics regulation. Obtained experimental data were analyzed using two models, assuming either rapid equilibrium between actomyosin and nucleotide or slow dissociation of ADP from actomyosin. We discuss the effect of mutations on the retention of the nucleotide by actomyosin and the potential role of electrostatic interactions in the Loop1 and in the force-generating region of human cardiac myosin on its regulation. We conclude that these salt bridges are essential for the proper functioning of beta isoform despite their absence in the alpha isoform.

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