ROLE OF INTRAMOLECULAR ELECTROSTATIC INTERACTIONS ON THE KINETICS OF HUMAN CARDIAC MYOSIN β -ISOFORM

Human cardiac myosin has two isoforms, α and β, sharing significant sequence similarity, but different in kinetics. Small differences in the sequence are responsible for distinct local inter-residue interactions within α and β isoforms, leading to such a dramatic difference in the rate of ADP release. Our analysis of structural kinetics of α and β isoforms using molecular dynamics simulations revealed distinct dynamics in SH1:SH2 helix region, loop 1 region, and loop I289-D324 region of myosin head. We identified permanent salt bridges in these regions on β-isoform, which are not present in the α-isoform. We hypothesized that the isoform-specific electrostatic interactions play a role in the difference of kinetic properties of myosin isoforms. We prepared R694N, E4Q, I303V:I313V,and D208Q:K450L mutants in the β-isoform background to destabilize electrostatic interactions in the proposed regions of the myosin head. We recombinantly expressed Wild type (WT) and the mutants of the human car- diac myosin head construct (1-843 amino acid residues) in differentiated C2C12 cells. Using the transient kinetics assays, we measured the kinetics of ADP release from actomyosin in the WT and mutant constructs of human cardiac myosin β-isoform. Mutant R694N showed faster rate of ADP release from actomyosin, compared to the wild type and other mutants, thus confirming that electrostatic interactions within the force-generating region of human cardiac myosin regulate ADP release and the duration of the strongly bound state of actomyosin.