Myosin Orientation in a Muscle Fiber using Bifunctional Spin Labels with 4 Degrees Angular Resolution

Abstract

We have measured the orientation of myosin light chain domain in a demembranated muscle fiber bundle using electron paramagnetic resonance (EPR) and a bifunctional spin label (BSL), with the angular resolution of 4 degrees in situ. A pair of (i,i+4) Cys residues was engineered on an alpha-helix of the regulatory light chain (RLC) on the N- and C- lobes. By exchanging endogenous RLC with mutant RLC labeled with BSL on oriented muscle fibers, we were able to resolve angular distributions in several biochemical states due to the stereospecific attachment of BSL's two disulfide bonds. In this setup, the accurate determination of BSL's angular coordinates allowed us to determine the orientation of individual structural elements with respect to the muscle fiber axis. The addition of ATP in the absence of Ca shifted the orientational distribution to a much more disordered one. By decorating demembranated fiber bundle with heavy meromyosin with RLC labeled with BSL, we observed different dynamics in the N- and C- lobe orientation. We compared these results with cardiac myosin. We also developed a new global fitting analysis to narrow the confidence intervals and enhance the sensitivity to azimuthal angle between pi orbital and magnetic field. While we find that the majority of the RLC helices are disordered even in the absence of ATP, we resolved an oriented population of myosin with lever arm being perpendicular to muscle fiber axis previously observed directly in insect flight muscle. This work was supported by NIH R01AR032961 and R37AG26160 to DDT. YS was supported by NIH T32AG29796 and University of Minnesota Interdisciplinary Doctoral Fellowship.