Fluorescence depolarization studies on the flexibility of myosin rod

Abstract

The single photon counting method was used to measure the decay of fluorescence polarization anisotropy of rabbit skeletal muscle myosin rods labeled with extrinsic fluorophores. Rods labeled with 8-anilino-1-naphthalenesulfonate (ANS) or 5-dimethylaminonaphthalene-1-sulfonyl chloride (DNS-Cl) exhibit negative rotational correlation times; the anisotropy increased with time. Possible artifactual causes for the negative decay times were ruled out. Such curves were expected for rigid rods when the fluorophore was bound so that the absorption and emission dipoles each made a small angle with the long axis of the molecule and lay on opposite sides of the rod. At pH 4 and below, rapid decay of the anisotropy (positive correlation times) indicated the presence of a freely bending region in the rod. This was probably the proteolytically sensitive region between light meromyosin and heavy meromyosin subfragment 2. At pH 8, no such free bending was observed, even at temperatures as high as 50.degree. C. From this observation and other physical properties of the rod, at pH 8, the hinge region had considerable resistance to bending. It was more like a spring than a free hinge. The rotational diffusion about the rod axis was faster than was predicted for a rigid, smooth molecule.