To understand muscle contraction at molecular level, we (i) identify in vivo structural changes that are associated with reaction steps within the actomyosin (cross-bridges) ATPase cycle; (ii) characterize the nature of these structural differences; and (iii) characterize mechanical properties of different actomyosin complexes in muscle. Under a wide range of conditions, response of the contractile system to osmotic compression can be used to screen for structural and mechanical differences of attached cross-bridges. In FY 92-93 we found that both the structure and elastic properties of the weakly attached cross-bridges are distinct from active crossbridges or cross-bridges in rigor. This is significant since the weakly attached cross-bridges represent the pre-force generating states. Our findings further strengthened the hypothesis that force generation results from a structural transformation from a weakly attached to a strongly attached configuration -- a hypothesis supported by the recently published crystal structures of actin, the myosin head and decorated actin filaments. Molecular structure of demembranated relaxed psoas muscle fibers at low temperature is being studied by two dimensional X-ray diffraction. Under these conditions, large fraction of cross-bridge are attached in the pre-force generating states. The diffraction patterns were found to consist of myosin layer lines without rigor features. The meridional intensities at 4400 nm and 2150 nm are approximately 5 times stronger that the 1430 nm reflection, a feature not observed previously in any other preparations. The present results suggest that attachment of pre- force generating cross-bridges shares few if any features with that of the cross-bridges in rigor and distribution of cross-bridges is repeatedly perturbed from the regularly spaced 143 nm levels along the thick filament. The significance of the finding is that even with the atomic structures of actin and myosin fragments becoming known, for any modelling of force generation in muscle, it will be necessary to have information of mass distribution along the filament axis such as those derived from in vivo two dimensional X-ray diffraction studies. Saturation of cross-bridges with nucleotide analogs ATP gamma S and GTP were found to vary over a wide range of concentrations. With modelling, these data should yield information on the elastic properties of a single attached myosin head, a central yet so far unknown quantity.
