Actin Residue Glu93 Is Identified as an Amino Acid Affecting Myosin Binding

• Published in: The Journal of biological chemistry Vol. 274; no. 40; pp. 28321 - 28328
• Main Authors: Razzaq, Azam, Schmitz, Stephan, Veigel, Claudia, Molloy, Justin E., Geeves, Michael A., Sparrow, John C.
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 01.10.1999; American Society for Biochemistry and Molecular Biology
• Subjects: actin; amino acid sequences; binding sites; Drosophila melanogaster; ionic strength; mutants; myosin; rabbits; skeletal muscle
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.274.40.28321

Many mutants have been described that affect the function of the actin encoded by the Drosophila melanogaster indirect flight muscle-specific actin gene,Act88F. We describe the development of procedures for purification of this actin from the other isoforms expressed in the fly as well as in vitro motility, single molecule force/displacement measurements, and stop-flow solution kinetic studies of the wild-type actin and that of the E93K mutation of theAct88F gene. We show that this mutation affects in vitro motility of F-actin, in both the presence and absence of methylcellulose, and the ability 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-actin are unchanged by the mutation. These results support the proposal (Holmes, K. C. (1995) Biophys J. 68, (suppl.) 2–7) that actin residue Glu93 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 site.