title: Fluorescence polarization transients from rhodamine isomers on the myosin regulatory light chain in skeletal muscle fibers.
abstract: Fluorescence polarization was used to examine orientation changes of two rhodamine probes bound to myosin heads in skeletal muscle fibers. Chicken gizzard myosin regulatory light chain (RLC) was labeled at Cys108 with either the 5- or the 6-isomer of iodoacetamidotetramethylrhodamine (IATR). Labeled RLC (termed Cys108-5 or Cys108-6) was exchanged for the endogenous RLC in single, skinned fibers from rabbit psoas muscle. Three independent fluorescence polarization ratios were used to determine the static angular distribution of the probe dipoles with respect to the fiber axis and the extent of probe motions on the nanosecond time scale of the fluorescence lifetime. We used step changes in fiber length to partially synchronize the transitions between biochemical, structural, and mechanical states of the myosin cross-bridges. Releases during active contraction tilted the Cys108-6 dipoles away from the fiber axis. This response saturated for releases beyond 3 nm/half-sarcomere (h.s.). Stretches in active contraction caused the dipoles 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 length changes are consistent with a partition of approximately 90% of the probes that did not tilt when length changes were applied and 10% of the probes that tilted. The responding fraction tilted approximately 30 degrees for a 7.5 nm/h.s. release and traversed the plane perpendicular to the fiber axis for larger releases. Stretches in rigor tilted Cys108-6 dipoles away from the fiber axis, which was the opposite of the response in active contraction. The transition from the rigor-type to the active-type response to stretch preceded the main force development when fibers were activated from rigor by photolysis of caged ATP in the presence of Ca2+. Polarization ratios for Cys108-6 in low ionic strength (20 mM) relaxing solution were compatible with a combination of the relaxed (200 mM ionic strength) and rigor intensities, but the response to length changes was of the active type. The nanosecond motions of the Cys108-6 dipole were restricted to a cone of approximately 20 degrees half-angle, and those of Cys108-5 dipole to a cone of approximately 25 degrees half-angle. These values changed little between relaxation, active contraction, and rigor. Cys108-5 showed very small-amplitude tilting toward the fiber axis for both stretches and releases in 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 the RLC undergoes a large angle change (approximately 60 degrees) between these 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 its own axis.
authors.affiliation: Pennsylvania Muscle Institute, University of Pennsylvania, Philadelphia 19104-6083, USA.
authors.forename: S C
authors.initials: SC
authors.lastname: Hopkins
authors.forename: C
authors.initials: C
authors.lastname: Sabido-David
authors.forename: J E
authors.initials: JE
authors.lastname: Corrie
authors.forename: M
authors.initials: M
authors.lastname: Irving
authors.forename: Y E
authors.initials: YE
authors.lastname: Goldman
mesh_terms: D000818:Animals; D002645:Chickens; D003545:Cysteine; D005454:Fluorescence Polarization / Q000295:instrumentation / Q000379:methods; D005456:Fluorescent Dyes; D005895:Gizzard, Avian; D066298:In Vitro Techniques; D007700:Kinetics; D008433:Mathematics; D008954:Models, Biological; D009119:Muscle Contraction / Q000502:physiology*; D009126:Muscle Relaxation; D018482:Muscle, Skeletal / Q000502:physiology*; D018995:Myosin Heavy Chains / Q000502:physiology; D018994:Myosin Light Chains / Q000032:analysis*; D011817:Rabbits; D012235:Rhodamines; D013997:Time Factors
chemical_list: D005456:Fluorescent Dyes; D018994:Myosin Light Chains; D012235:Rhodamines; C034996:tetramethylrhodamine iodoacetamide; D018995:Myosin Heavy Chains; D003545:Cysteine