Unusual Structure and Function of Active Intermediate of Myosin Realized during Sliding Movement : A Possibility for Energy Storage State

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As a means to investigate the 3-D structural changes of sliding actomyosin, we have developed a "differential pattern-recognition method" to quantitatively compare the surface profiles of the protein assembly instantaneously captured. by quick-freeze deep-etch replication, with those of the artificial images simulated from the atomic models of the target. We discovered that actin-attached myosin head during sliding takes a configuration kinked to the opposite direction from widely believed ATP-bound form. Since we also found the presence of particles in the same configuration but with different attachment angle to actin, we confirmed the significance of that novel structure, and proposed a new scheme of crossbridge movement, that is quite different from the conventional one, but comprehensively accounts for the presence of all the observed structures. We also analyzed the structures of actin-attached myosin-V motor-domain with long lever-arm moiety, during processive walking movement a … More long actin-filament, by a similar means. The trail head showed a rigor-like pattern as postulated in a preceding study, but the images of the lead heads matched by far better with our novel structure than ATP-bound form postulated in the above study, suggesting that the structural change we found in myosin-II might be universally applicable to unconventional myosin species. We are also developing a novel multifunctional microscopic probe to indicate the localization of specific sites or subdomains in the protein assembly, which might enable us to follow the dynamic behavior of given molecules even within intracellular environments. We successfully fused the probe construct to myosin head and attempted to visualize the movement of the probe using the world best-rated atomic force microscope with highest temporal and spatial resolutions. However, the movement was too fast and subtle and the material was not well-immobilized onto mica surface. We would retry after enhancing the adsorption and introducing multiple probes in a molecule. Less