MUTATIONAL ANALYSIS OF THE ROLE OF HYDROPHOBIC RESIDUES IN THE 338-348-HELIX ON ACTIN IN ACTOMYOSIN INTERACTIONS

Yeast actin mutants with alanines replacing 1341 and 1345 were studied to assess the role of hydrophobic residues in the alpha-helix 338-348 in interactions with myosin. In structural models of the actomyosin c omplex, this helix on actin was assigned a prominent role in the stron g binding of myosin to actin. Substitution of 1341 with alanine reduce d the strong binding of actin to myosin subfragment-l (S1) 9-fold comp ared to wild-type actin. In addition, the V-max of the actin-activated S1 ATPase was reduced 4-fold with no change in the K-m, In contrast, substitution of I345 with alanine had no significant effect on either the strong binding to S1 or the actin activation of S1 ATPase, The I34 1A actin filaments were found to slide in the in vitro motility assays at a lower mean velocity (1.6 +/- 0.4 mu m/s) than wildtype actin fil aments (2.6 +/- 0.3 mu m/s). Only 65% of the mutant actin filaments mo ved in such assays in comparison to 95% of the wild-type filaments, Ho wever, addition of 2.0 mM MgADP to the motility assay buffer induced m ovement of all the I341A filaments at a velocity (1.6 +/- 0.1 mu m/s) similar to that of wild-type actin (1.7 +/- 0.1 mu m/s). The decrease in motility of the I341A actin filaments in the absence of ADP was att ributed to a negative load slowing the mutant filaments and the smalle r force produced by the heavy meromyosin and I341A actin system. The l atter conclusion was confirmed by showing that a greater percentage of NEM-modified heavy meromyosin (external load) was required for arrest ing the motion of wild-type actin in the in vitro motility assay than that needed for stopping the I341A filaments.