Cooperativity in Actin Filament and Its Functional Implication : Structural analyses by Quick-Freeze Deep-Etch Replica Electron Microscopy

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This study aimed to capture the instantaneous structure of sliding actomyosin by quick-freeze deep-etch electron microscopy and to analyze the three-dimensional structural changes not only of crossbridges but also of actin filaments which had been postulated to work as simple track of myosin-motor movement. After completion of innovative methodology for 3-D reconstruction of replica specimens, we started the application of similar process to the reconstruction of bulky solid structure like negatively stained specimens. We also devised another methodology to predict the expected deep-etch replica image of the protein molecule, utilizing its atomic coordinates. The structure of myosin heads in rigor-complex matched pretty well to the model proposed by docking the crystal structure of actin and myosin S1. However, most of the crossbridges under sliding condition, unlike prediction, exhibited the configuration kinked to the opposite direction from that of ATP-bound ones. Since the observed … More structure appeared very similar to that of pPDM-crosslinked myosin/ADP complex, we compared quantitatively the surface profile of replica images with that of simulated model-images, utilizing new pattern-matching procedure. The result clearly confirmed that myosin head is oppositely kinked during sliding. This conflicts conventional "Tilting lever-arm hypothesis" and we must consider the new mechanism incorporating the existence of the novel structure.Actin filaments easily cut into small pieces during in vitro sliding. We compared the actual replica images of such particles with the images simulated from Holmes model of actin, and found that the phase of actin filaments' helical structure was interrupted by myosin binding. It is known that actin filaments are fragile by the application of a torque-force. The possibility is that such torque force may apply as an intrinsic and mandatory mechanical process for sliding. We would propose the new hypothesis of actomyosin sliding mechanism that might implicate all the observed phenomena in microscopic fields. Less