ROTATION OF MYOSIN CROSS-BRIDGES IN SKELETAL MUSCLES

In a classical hypothesis of muscle contraction, the cyclic reorientations of myosin cross-bridges are the cause of contraction. However, this idea has recently been contested by the Electron Paramagnetic Resonance results which suggest that there exists only a single orientation of myosin. It is therefore essential to test, by alternative techniques, for the presence of different orientations of myosin. To this end, it is proposed to a) develop a method of measuring the spatial attitude of the isolated actomyosin in a gel, and b) test for the presence of the rotational motions of the cross-bridges by perturbation techniques. All experiments require a laser source and a microscope to determine cross-bridge orientation by measuring polarization of fluorescence of myosin-bound dye. In project 1, a new technique of measuring the orientation of isolated proteins oriented by gel electrophoresis is described, and it is proposed to apply this technique to measure the attitude of isolated myosin subfragment- 1 (S-1) with respect of F-actin during different steps in a sequence of intermediate states of actin-activated cycle of ATPase activity of S-1. In project 2, it is proposed to rapidly perturb a steady state population of active cross-bridges by depopulating a narrow angular sector of cross-bridges by a powerful polarized light pulse; the repopulation of this sector will provide the information about the rate of anticipated cross-bridge reorientations and will be followed by polarization methods. In project 3, it is proposed to use the wavelength-dependent polarization of fluorescence method in order to establish whether the angular reorientations induced by the addition of MgADP are a universal property of the cross-bridges, or whether they are observable only for specific probes. In project 4, it is proposed to use laser photolysis method to rapidly produce millimolar concentration of ATP by photolysis of photolysis "caged" precursor of ATP previously diffused into muscle. This is expected to activate all cross-bridges simultaneously, and their rotational motion, if present, will be determined by polarization of fluorescence method. The health relatedness of these projects are that the information about the mechanism muscle contraction will ultimately lead to the positive intervention in diseases of muscle.