MODULATION OF ACTIN AFFINITY AND ACTOMYOSIN ADENOSINE-TRIPHOSPHATASE BY CHARGE CHANGES IN THE MYOSIN MOTOR DOMAIN

The effects of mutations in an actin-binding surface loop of myosin (l oop 2) are described. Part of loop 2, the segment between myosin resid ues 618 and 622, was replaced with sequences enlarged by the introduct ion of positively charged GKK or neutral GNN motifs. Constructs with l oops carrying up to 20 additional amino acids and charge variations fr om -1 to +12 were produced. Steady-state and transient kinetics were u sed to characterize the enzymatic behavior of the mutant motor domains . Binding of nucleotide was not affected by any of the alterations in loop 2. In regard to their interaction with actin, constructs with mod erate charge changes (-1 to +2) displayed wild-type-like behavior. Int roduction of more than one GKK motif led to stronger coupling between the actin-and nucleotide-binding sites of myosin and an up to 1000-fol d increased affinity for actin in the absence of ATP and at zero ionic strength, In comparison to the wild-type construct M765, constructs w ith 4-12 extra charges displayed an increased dependence on ionic stre ngth in their interaction with actin, a 2-3-fold increase in k(cat), a more than 10-fold reduction in K-app for actin, and a 34-70-fold incr ease in catalytic efficiency.