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Single Molecule Fluorescence Polarization of Calmodulin in Myosin VI


PIs: Yujie Sun, Harry W. Schroeder, John F. Beausang, Kazuaki Homma, Mitsuo Ikebe, Yale E. Goldman

Myosin VI is a double-headed motor protein involved in membrane traffic and cell migration that moves processively towards pointed ends of actin filaments. The step size of myosin VI (20-40 nm) is surprisingly large considering its short neck domain containing only 1 or 2 calmodulins (CaMs). Single fluorophore tracking has shown that myosin VI moves by a hand-over-hand mechanism. To deter-mine orientation changes in the CaM-binding neck during processive motility, we measured single molecule fluorescence polarization (SMFP) of bifunctional rhoda-mine-labeled CaM exchanged into myosin VI.

Results and Conclusions

Unlike myosin V, the motility of myosin VI was not necessarily accompanied by abrupt changes of fluorophore angle () relative to the axis of actin. The azimuthal angle () of the lever arm label often showed abrupt changes indicating twisting of the myosin VI around the actin axis and large azimuthal flexibility (the histogram shows twist amplitudes). The rate of these tilts increased with [ATP]. These azimuthal changes and their variability suggest a mechanism in which myosin VI can search for an actin binding site almost ±90° around the filament axis. This result may rationalize the puzzling discrepancy between step size and helical path of this molecular motor.

Reference: Beausang, J.F., Schroeder, H.W., III, Gilmour, J.A., and Goldman, Y.E. Twirling of Actin by Myosins II and V. Biophys J., 90:587a. 2006.



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