Structural transition at actin's N-terminus in the actomyosin cross-bridgecycle

Force and motion generation by actomyosin involves the cyclic formation and transition between weakly and strongly bound complexes of these proteins. Actin's N-terminus is believed to play a greater role in the formation of t he weakly bound actomyosin states than in the formation of the strongly bou nd actomyosin states. II has been the goal of this project to determine whe ther the interaction of actin's N-terminus with myosin changes upon transit ion between these two states. To this end, a yeast actin mutant, Cys-1, was constructed by the insertion of a cysteine residue at actin's N-terminus a nd replacement of the C-terminal cysteine with alanine. The N-terminal cyst eine was labeled stoichiometrically with pyrene maleimide, and the properti es of the modified mutant actin were examined prior to spectroscopic measur ements. Among these properties, actin polymerization, strong S1 binding, an d the activation of S1 ATPase by pyrenyl-Cys-1 actin were not significantly different from those of wild-type yeast: actin, while small changes were o bserved in the weak S1 binding and the in vitro motility of actin filaments . Fluorescence changes upon binding of S1 to pyrenyl-Cys-1 actin were measu red for the strongly (with or without ADP) and weakly (with ATP and ATP gam ma S) bound acto-S1 states. The fluorescence increased in each case, but th e increase was greater (by about 75%) in the presence of MgATP and MgATP ga mma S than in the rigor state. This demonstrates a transition at the SI con tact with actin's N-terminus between the weakly and strongly bound states, and implies either a closer proximity of the pyrene probe on Cys-1 to struc tural elements on S1 (most likely the loop of residues 626-647) or greater S1-induced changes at the N-terminus of actin in the weakly bound acto-S1 s tates.