Polarized Fluorescence Depletion Reports Orientation Distribution and Rotational Dynamics of Muscle Cross-Bridges

Abstract

The method of polarized fluorescence depletion (PFD) has been applied to enhance the resolution of orientational distributions and dynamics obtained from fluorescence polarization (FP) experiments on ordered systems, particularly in muscle fibers. Previous FP data from single fluorescent probes were limited to the 2(nd)- and 4(th)-rank order parameters, <P(2)(cos beta)> and <P(4)(cos beta)>, of the probe angular distribution (beta) relative to the fiber axis and <P(2d)>, a coefficient describing the extent of rapid probe motions. We applied intense 12-micros polarized photoselection pulses to transiently populate the triplet state of rhodamine probes and measured the polarization of the ground-state depletion using a weak interrogation beam. PFD provides dynamic information describing the extent of motions on the time scale between the fluorescence lifetime (e.g., 4 ns) and the duration of the photoselection pulse and it potentially supplies information about the probe angular distribution corresponding to order parameters above rank 4. Gizzard myosin regulatory light chain (RLC) was labeled with the 6-isomer of iodoacetamidotetramethylrhodamine and exchanged into rabbit psoas muscle fibers. In active contraction, dynamic motions of the RLC on the PFD time scale were intermediate between those observed in relaxation and rigor. The results indicate that previously observed disorder of the light chain region in contraction can be ascribed principally to dynamic motions on the microsecond time scale.

Structural changes in troponin in response to Ca 2+ and myosin binding to thin filaments during activation of skeletal muscle

“…The intensities of the resulting four components were measured by photomultiplier tubes (R4632; Hamamatsu Photonics). The relative transmittances of the excitation and emission optics and photomultiplier sensitivities were determined by using an isotropic film of rhodamine in poly(vinyl alcohol) sandwiched between 45°prisms (38).…”

Toward Protein Structure In Situ: Comparison of Two Bifunctional Rhodamine Adducts of Troponin C

“…The resultant polarized fluorescence intensities were measured simultaneously by four photomultipliers (R4632, Hamamatsu, Hamamatsu City, Japan) for each excitation polarization. Relative transmittances of the excitation and emission channels and photomultiplier sensitivities were measured using an isotropic film of rhodamine in polyvinylalcohol sandwiched between two 45°prisms (18), and appropriate correction factors applied to the fiber data. Three independent order parameters, AEP 2d ae, AEP 2 ae, and AEP 4 ae, were calculated from the eight corrected intensities (19).…”

Bifunctional Rhodamine Probes of Myosin Regulatory Light Chain Orientation in Relaxed Skeletal Muscle Fibers

“…The resolution of ME analysis could probably be improved in future studies by using more probe attachment sites (van der Heide et al, 2000), or by measuring higher-rank order parameters (hP 6 i, hP 8 i, etc.) by pulsed fluorescence excitation techniques (Bell et al, 2002).…”

“…Third, changes in the azimuthal orientation distribution of the probes around the symmetry axis cannot usually be deduced from steady-state polarized fluorescence measurements. Some of these limitations may be removed by further developments of the technique (van der Heide et al, 2000;Bell et al, 2002).…”