FLUORESCENCE POLARIZATION TRANSIENTS FROM RHODAMINE ISOMERS ON THE MYOSIN REGULATORY LIGHT-CHAIN IN SKELETAL-MUSCLE FIBERS

Fluorescence polarization was used to examine orientation changes of t wo rhodamine probes bound to myosin heads in skeletal muscle fibers. C hicken gizzard myosin regulatory light chain (RLC) was labeled at Cys( 108) with either the 5- or the 6-isomer of iodoacetamidotetramethylrho damine (IATR). Labeled RLC (termed Cys(108-5) or Cys(108-6)) was excha nged for the endogenous RLC in single, skinned fibers from rabbit psoa s muscle. Three independent fluorescence polarization ratios were used to determine the static angular distribution of the probe dipoles wit h respect to the fiber axis and the extent of probe motions on the nan osecond time scale of the fluorescence lifetime. We used step changes in fiber length to partially synchronize the transitions between bioch emical, structural, and mechanical states of the myosin cross-bridges. Releases during active contraction tilted the Cys(108-6) dipoles away from the fiber axis. This response saturated for releases beyond 3 nm /half-sarcomere (h.s.). Stretches in active contraction caused the dip oles to tilt toward the fiber axis, with no evidence of saturation for stretches up to 7 nm/h.s. These nonlinearities of the response to len gth changes are consistent with a partition of similar to 90% of the p robes that did not tilt when length changes were applied and 10% of th e probes that tilted. The responding fraction tilted similar to 30 deg rees for a 7.5 nm/h.s. release and traversed the plane perpendicular t o the fiber axis for larger releases. Stretches in rigor tilted Cys(10 8-6) dipoles away from the fiber axis, which was the opposite of the r esponse in active contraction. The transition from the rigor-type to t he active-type response to stretch preceded the main force development when fibers were activated from rigor by photolysis of caged ATP in t he presence of Ca2+. Polarization ratios for Cys(108-6) in low ionic s trength (20 mM) relaxing solution were compatible with a combination o f the relaxed (200 mM ionic strength) and rigor intensities, but the r esponse to length changes was of the active type. The nanosecond motio ns of the Cys(108-6) dipole were restricted to a cone of similar to 20 degrees half-angle, and those of Cys(108-5) dipole to a cone of simil ar to 25 degrees half-angle. These values changed little between relax ation, active contraction, and rigor. Cys(108-5) showed very small-amp litude tilting toward the fiber axis for both stretches and releases i n active contraction, but much larger amplitude tilting in rigor. The marked differences in these responses to length steps between the two probe isomers and between active contraction and rigor suggest that th e RLC undergoes a large angle change (similar to 60 degrees) between t hese two states. This motion is likely to be a combination of tilting of the RLC relative to the fiber axis and twisting of the RLC about it s own axis.