Project Details


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.
Effective start/end date1/04/9631/08/96