|Budget Amount *help
¥29,400,000 (Direct Cost : ¥29,400,000)
Fiscal Year 1996 : ¥3,000,000 (Direct Cost : ¥3,000,000)
Fiscal Year 1995 : ¥9,700,000 (Direct Cost : ¥9,700,000)
Fiscal Year 1994 : ¥16,700,000 (Direct Cost : ¥16,700,000)
We have constructed a myosin motor domain consisted of the heavy chain alone(760 residues), which retained motor functions. By using this construct, we succeeded to crystallize the myosin motor domain, and solve its structure at the atomic resolution (2.7A). By using this structural information, we carried out carried out site-directed mutagenesis of functionally important regions of myosin, especially the switch I and switch II loops. A loop comprising residues 454-459 of Dictyostelium myosin II is structurally and functionally equivalent to the switch II loop of the G-protein family. The consensus sequence of the switch II loop of the myosin family is DIXGFE.In order to determine the functions of each of the conserved residues, alanine scanning mutagenesis was carried out on the dictyostelium myosin II heavy chain gene. Examination of in vivo and in vitro motor functions of the mutant myosins revealed that the I455A and S456A mutants retained those functions while the D454A,G457A,F458A and E459A mutants lost them. Biochemical analysis of the latter myosins showed that the G457A and E459A mutants lost the basal ATPase activity by blocking of the isomerization and hydrolysis speps of the ATPase cycle, respectively. The F458A mutant, however, lost the actin-activated ATPase activity without loss of the basal ATPase activity. These results are discussed in terms of the crystal structure of the Dictyostelium myosin motor domain.