The central goal of our research is to understand the mechanism of muscle contraction by pursuing in-depth studies of molecular structure and physiological functions of skeletal muscle. Muscle is a tissue which is specialized for motility but similar motile processes occur in virtually all eukaryotic cells. While our understanding of muscle contraction is improving, the basic force production mechanism is still not yet fully understood nor is the mechanism of its regulation. Our research efforts have been devoted to determine structural changes in the acto-myosin (attached cross-bridge) complex during ATP hydrolysis. The general approach is to record X-ray diffraction patterns from fully functioning demembranated muscle fibers under physiological or near physiological conditions. High resolution X-ray diffraction patterns from relaxed muscle fibers obtained earlier were analyzed in detail during last year. Analysis of intensity distributions and background levels of the diffraction patterns suggests that orientations of the weakly attached cross-bridges are random as if the cross-bridges were free (detached). It appears that cross-bridges, in their cyclic interaction with actin, first bind to actin with low affinity and with random (non-stereospecific) orientations. A current working hypothesis proposed by us (Brenner et al., 1995) is that a subsequent transition from the non-stereospecific weak binding to a rigid strong binding results in isometric force generation. The present results provide a critical step in confirming the hypothesis. Significant progress was made in modelling the filament structures in rabbit (mammalian) muscle cells. Modelling suggests that at physiological temperature the cross-bridges form a helix wrapping around and close to the surface of the myosin filament backbone. At low temperature (4 degrees C), the fraction of helically ordered cross-bridges is reduced by about one half and the center of mass of the cross-bridges moves out. In addition, it appears that within a relaxed muscle cell, there exists an equilibrium between a helically ordered population and a disordered population of cross-bridges; the equilibrium is primarily a function of temperature. Within the disordered population, however, there is an equilibrium between the weakly attached and the detached cross-bridges and this equilibrium is mostly sensitive to ionic strength. The existence of an equilibrium between ordered and disordered populations in muscle could prove to be critical for the activation process of muscle contraction. For the past twenty years, the nucleotide analog AMP-PNP (adenylyl imido diphosphate) has been widely thought to cause strong binding between mysoin and actin, simulating the force generating state in the cross-bridge cycle. We discovered, however, cross-bridges saturated with AMP-PNP behave as weak binding analogs. Binding studies in solution confirmed the results obtained in muscle fibers, indicating that AMP-PNP is an ATP analog.
