Title: Instability of the S1/S2 Hinge is not Affected by the Myosin Filament
Abstract: The S1/S2 hinge of myosin is thought to be unstable in some species of myosin such as myosin VI and certain scallop muscle myosin. To examine the stability of the S1/S2 hinge of rabbit skeletal muscle myosin, gravitational force spectrometry was used to measure the separation of myosin heads bound in rigor to actin immobilized on glass microspheres. The molecular lengths of this separation were measured at different levels of applied force up to the limit that the rigor bond would sustain of about 10 pN in these experiments. The molecular lengths were found to increase linearly with the applied force up to lengths exceeding 100 nm which requires a partial unraveling of the S2 coiled coil in the single myosin molecule. Free-fall force spectrometry on such single myosin molecules confirmed that the coiled coil could reversibly unravel and refold at forces less than 13 pN. In order to test whether thick filament formation would affect this instability under force, synthetic cofilaments of proteolytically isolated myosin rod and intact myosin were prepared at different ratios of myosin to rod up to ratios where it is expected that only one intact myosin would be present per myosin rod cofilament. The force-distance relationships for myosin in the cofilament were not substantially different from those of single myosin molecules. These data indicate the possibility that vertebrate skeletal muscle myosin may also utilize S1/S2 hinge unraveling to provide flexibility during crossbridge formation so that a productive binding orientation is achieved. The reversibility of the unraveling of the coiled coil might contribute to the mechanism of motility under some circumstances as has been proposed for non-muscle myosin VI.