Orientation and motion of the myosin regulatory light chain region in skeletal muscle fibers during force generation and filament sliding

The orientation of the regulatory light chain (RLC) binding region in myosin was investigated by measuring fluorescence polarization from rhodamine probes in muscle fibers during events of the contractile cycle. Each of 12 differently labeled RLCs were exchanged for native RLC in skinned rabbit psoas muscle fibers. Three independent fluorescence polarization ratios were measured at 50 $\mu$s time resolution to determine parameters of the orientational distributions for these probe dipoles with respect to the fiber\u27s axis. The probes were observed to tilt during and after imposed length steps in active contraction indicating tilting motions of the RLC during filament sliding and force generation. These probes were observed to tilt during imposed stretches on rigor fibers as well. In the case of the 6-isomer bound to Cys 108, the direction of tilting during stretches in rigor was opposite to that of active contraction, suggesting the dipoles tilt across a plane 90$\sp\circ$ to the fiber\u27s axis between these states. The time course of this tilting was monitored by sinusoidal length oscillations at 500 Hz during activation from rigor by photolysis of caged ATP. The amount of tilting was a non-linear function of step size. The tilting was consistent with some portion of the total distribution dipoles crossing the 90$\sp\circ$ plane with respect to the fiber\u27s axis during their working strokes. The direction of tilting observed from the whole group of probes suggested that the RLC moves toward its rigor orientation during filament sliding and force generation and back toward its active orientation during a stretch imposed in rigor. The orientation of the RLC itself was determined by measuring polarization ratios from a series of three bifunctional probe dipoles, where the disposition of each dipole relative to the RLC crystal structure was predetermined by its attachment to appropriately engineered pairs of cysteines in chicken gizzard RLC. The RLC was observed to undergo a large twist between active and rigor, and the motions of the RLC during length changes suggest that this twist occurs during the working stroke of myosin