In the lever arm model for actomyosin motility, the only roles of actin filaments are to stimulate Pi release from myosin and to provide foothold for tension generation. However, actin filaments undergo myosin-dependent cooperative conformational changes. To understand the possible functions of those changes in actomyosin motility, we hoped to obtain mutant actins with defective conformational changes, and consequently, impaired motility. Mutant actins of this class are presumably dominantly inhibitory by impairing functions of copolymerized wild type actin. Thus, we constructed a series of mutant genes in which Gly residues in actin were systematically substituted to Val, and identified 5 dominant negative mutant actins on the basis of growth inhibition when expressed in yeast. Of these, we chose G146V mutant for further analyses, because changing Gly146 at the hinge between the small and large domains of actin might impair relative conformational changes between the two domains. G146V actin polymerized more readily than wt, but gliding velocity and force production of G146V filaments on skeletal (sk) HMM surfaces decreased by ∼80%. Kinetic analyses indicated that prolonged strongly-bound state is not the cause of the slow movement. In contrast, G146V filaments moved and produced force normally on myosin V. To probe structural changes of actin involving Gly146, we measured FRET efficiency between two fluorophores in the small and large domains (Thr41 and Ala235) of individual actin molecules in filaments. Control actin subunits take at least two different states, while most of the G146V actin subunits were in one state with a higher FRET efficiency. These results suggest the possibility that G146V actin take inappropriate conformation for motility of sk myosin. We are currently performing FRET experiments in the presence of sk HMM and myosin V.