Myosin head rotation in muscle fibers measured using polarized fluorescence photobleaching recovery.
The technique of polarized fluorescence photobleaching recovery (PFPR) has been applied for the first time to investigation of the rotational correlation time of the myosin head in muscle fibers. This is a novel application of PFPR because it is the first time PFPR has been applied to a sample which is not cylindrically symmetric about the optical axis. Therefore we present a method for analysis of PFPR results from an oriented sample such as the muscle fibers aligned perpendicularly to the optical axis used here. Control experiments performed on fluorescently labeled myosin heads in solution demonstrate that, under some conditions, our PFPR apparatus can easily measure a rotational correlation time of less than 200 μs. Validity of this application of PFPR to muscle fibers is provided by the agreement of our results with published results from a variety of other spectroscopic techniques. In particular, using glycerinated rabbit psoas muscle fibers, we find that for relaxed fibers and isometrically contracting fibers, the myosin heads undergo high-amplitude rotations on the submillisecond time domain. For fibers in rigor the myosin heads are highly oriented and nearly immobile. For fibers in ADP the myosin heads are highly ordered in a distribution quite different from that in rigor, and they are slightly more mobile than in rigor.
Department of Biochemistry and Molecular Biology, Mayo Foundation, 55905, Rochester, Minnesota, burghardt@mayo.edu.
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