Myosin Regulatory Domain Orientation in Skeletal Muscle Fibers: Application of Novel Electron Paramagnetic Resonance Spectral Decomposition and Molecular Modeling Methods

Abstract

Reorientation of the regulatory domain of the myosin head is a feature of all current models of force generation in muscle. We have determined the orientation of the myosin regulatory light chain (RLC) using a spin-label bound rigidly and stereospecifically to the single Cys-154 of a mutant skeletal isoform. Labeled RLC was reconstituted into skeletal muscle fibers using a modified method that results in near-stoichiometric levels of RLC and fully functional muscle. Complex electron paramagnetic resonance spectra obtained in rigor necessitated the development of a novel decomposition technique. The strength of this method is that no specific model for a complex orientational distribution was presumed. The global analysis of a series of spectra, from fibers tilted with respect to the magnetic field, revealed two populations: one well-ordered (+/-15 degrees ) with the spin-label z axis parallel to actin, and a second population with a large distribution (+/-60 degrees ). A lack of order in relaxed or nonoverlap fibers demonstrated that regulatory domain ordering was defined by interaction with actin rather than the thick filament surface. No order was observed in the regulatory domain during isometric contraction, consistent with the substantial reorientation that occurs during force generation. For the first time, spin-label orientation has been interpreted in terms of the orientation of a labeled domain. A Monte Carlo conformational search technique was used to determine the orientation of the spin-label with respect to the protein. This in turn allows determination of the absolute orientation of the regulatory domain with respect to the actin axis. The comparison with the electron microscopy reconstructions verified the accuracy of the method; the electron paramagnetic resonance determined that axial orientation was within 10 degrees of the electron microscopy model.

Myosin and Other Energy-Transducing ATPases: Structural Dynamics Studied by Electron Paramagnetic Resonance

“…The spin label at the RLC of intrinsic myosin heads appeared considerable disordered in muscle fibers in the rigor (−ATP) state, with the corresponding spectra similar to that of random orientation (Figure 2A) (Arata,). Similar results were obtained by other laboratories [40][41][42][43][44][45]. However, the difference spectra between rigor and relaxed (+ATP, −Ca 2+ ) spectra demonstrated two kinds of well-ordered spectral components.…”

Poorly Understood Aspects of Striated Muscle Contraction

“…The subsequent, critical step in the force-generating cycle is the binding of the myosin head to the actin filament, forming a so-called cross-bridge. The initial binding is nonspecific [ 69 , 70 ] and dynamically disordered with a range of azimuthal and axial angles of both motor domain and the light chain binding lever arm [ 71 – 73 ] relative to the actin filament. Furthermore, this initial weak binding is mainly electrostatic in nature [ 69 , 70 ] with attached and detached states in rapid equilibrium.…”

Structure and Dynamics of the Force-Generating Domain of Myosin Probed by Multifrequency Electron Paramagnetic Resonance

“…The spectrum typically depends less on peptide backbone motion than on restriction of a spin label's motion by adjacent structural elements of a protein (35). Careful interpretation of the EPR spectral lineshape in terms of the spin-label orientational distribution is important for a precise distance determination in DEER (36,37), to find relative orientation of protein domains (38,39) and for a rigorous analysis of multicomponent spectra. In our study, we emphasize the role of multifrequency EPR in spectral analysis, which has allowed us to resolve multiple structural states of the force-generating domain of myosin in S1.ADP.V i biochemical state.…”