Actin residue Glu(93) is identified as an amino acid affecting myosin binding

Many mutants have been described that affect the function of the actin enco ded by the Drosophila melanogaster indirect flight muscle-specific actin ge ne, Act88F. We describe the development of procedures for purification of t his actin from the other isoforms expressed in the fly as well as in vitro motility, single molecule force/displacement measurements, and stop-flow so lution kinetic studies of the wild-type actin and that of the E93K mutation of the Act88F gene. We show that this mutation affects in vitro motility o f F-actin, in both the presence and absence of methylcellulose, and the abi lity of the ACT88F actin to bind the S1 fragment of rabbit skeletal myosin, However, optical tweezer measurements of the actomyosin working stroke and the force transmitted from the rabbit heavy meromyosin to and through F-ac tin are unchanged by the mutation. These results support the proposal (Holm es, K. C, (1995) Biophys J. 68, (suppl,) 2-7) that actin residue Glu(93) is part of the secondary myosin binding site and suggest that myosin binding occurs first at the primary myosin binding site and then at the secondary s ite.