Polarized fluorescence depletion reports orientation distribution and rotational dynamics of muscle cross -bridges.

摘要

A novel method has been developed to obtain high resolution orientational distributions and dynamics from fluorescence polarization (FT) experiments in muscle fibers. Gizzard myosin regulatory light chain (RLC) was labeled with the 6-isomer of IATR (Ling et al., 1996) and exchanged into rabbit psoas muscle fibers. Previous FP data from single probes were limited to the 2 nd- and 4th-rank order parameters, ⟨P 2(cosbeta)⟩ and ⟨P4 (cosbeta)⟩, of the probe angular distribution (beta) relative to the fiber axis and ⟨P2d⟩, a coefficient describing extent of rapid probe motions (Dale et al., 1999). We applied intense 12 mus photoselection pulses to transiently populate the probe triplet state and measured polarization of the ground state depletion using a weak interrogation beam. Transient photodepletion provides information about the quasi-static angular distribution up to ⟨P6(cosbeta)⟩ as well as dynamic information embodied in ⟨P2p ⟩ and ⟨P4p⟩, which describe the extent that ⟨P2⟩ and ⟨P4⟩ are reduced due to motions on timescales longer than the fluorescence lifetime but shorter than the photoselection pulse. In active contraction, RLC motions were intermediate between relaxed and rigor. These results indicate that the previously observed disorder of the light chain region in contraction can largely be ascribed to dynamic motions.