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Biophys J, August 2002, p. 1050-1073, Vol. 83, No. 2

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

Marcus G. Bell,* Robert E. Dale,*dagger Uulke A. van der Heide,*Dagger and Yale E. Goldman*

 *Pennsylvania Muscle Institute, The School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6083;  dagger King's College London, London SE1 1UL, England; and  Dagger Universitair Medisch Centrum Utrecht, The Netherlands

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 2nd- and 4th-rank order parameters, < P2(cos beta )> and < P4(cos beta )> , of the probe angular distribution (beta ) relative to the fiber axis and < P2d> , a coefficient describing the extent of rapid probe motions. We applied intense 12-µs 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.

Biophys J, August 2002, p. 1050-1073, Vol. 83, No. 2
© 2002 by the Biophysical Society   0006-3495/02/08/1050/24  $2.00


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