Measuring Myosin Light Chain Domain Orientation in the Pre-Power Stroke AlF4 States with a Bifunctional Spin Label in Skinned Muscle Fibers

Abstract

We are using electron paramagnetic resonance and site-directed spin labeling to measure the orientation of the light chain domain of skeletal muscle myosin in pre-power stroke states trapped by the phosphate analog AlF4. Previous work (Kraft et al. (2005) Proc. Natl. Acad. Sci. USA 102:13861-66) has shown via single-fiber X-ray diffraction that AlF4 traps two distinct pre-powerstroke myosin states in activated muscle. The first state (ADP.AlF4-I) produces a weak actin binding, disordered myosin whereas the second state (ADP.AlF4-II) produces a strong binding, stereospecific actomyosin complex. This weak-to-strong transition is a clear indication of the initiation of the power stroke before phosphate release, but it does not reveal the state of the light chain domain (LCD), which undergoes a rotation during the power stroke. We measured the orientation of the light chain domain by exchanging the native regulatory light chain (RLC) of skinned rabbit psoas muscle fiber bundles with a Di-Cys mutant RLC labeled with a bifunctional spin label. Our group has shown previously (Thompson and Naber et al. 2008 Biophys J, in press) that a bifunctional methanethiosulfonate spin label binds rigidly to myosin and reports protein orientation accurately. EPR spectra of oriented fibers show that the LCD produces a distinct orientation from rigor and relaxation in the ADP.AlF4-II state whereas the ADP.AlF4-I state is indistinguishable from relaxed muscle, supporting the hypothesis that the II state represents a state early in the power stroke with a distinct LCD orientation. This work was supported by NIH (AR32961, AR007612). We thank Bernhard Brenner and Theresia Kraft for guidance.