Structure and dynamics of the force-generating domain of myosin probed by multifrequency electron paramagnetic resonance.
Spin-labeling and multifrequency EPR spectroscopy were used to probe the dynamic local structure of skeletal myosin in the region of force generation. Subfragment 1 (S1) of rabbit skeletal myosin was labeled with an iodoacetamide spin label at C707 (SH1). X- and W-band EPR spectra were recorded for the apo state and in the presence of ADP and nucleotide analogs. EPR spectra were analyzed in terms of spin-label rotational motion within myosin by fitting them with simulated spectra. Two models were considered: rapid-limit oscillation (spectrum-dependent on the orientational distribution only) and slow restricted motion (spectrum-dependent on the rotational correlation time and the orientational distribution). The global analysis of spectra obtained at two microwave frequencies (9.4 GHz and 94 GHz) produced clear support for the second model and enabled detailed determination of rates and amplitudes of rotational motion and resolution of multiple conformational states. The apo biochemical state is well-described by a single structural state of myosin (M) with very restricted slow motion of the spin label. The ADP-bound biochemical state of myosin also reveals a single structural state (M*, shown previously to be the same as the post-powerstroke ATP-bound state), with less restricted slow motion of the spin label. In contrast, the extra resolution available at 94 GHz reveals that the EPR spectrum of the S1.ADP.V(i)-bound biochemical state of myosin, which presumably mimics the S1.ADP.P(i) state, is resolved clearly into three spectral components (structural states). One state is indistinguishable from that of the ADP-bound state (M*) and is characterized by moderate restriction and slow motion, with a mole fraction of 16%. The remaining 84% (M**) contains two additional components and is characterized by fast rotation about the x axis of the spin label. After analyzing EPR spectra, myosin ATPase activity, and available structural information for myosin II, we conclude that post-powerstroke and pre-powerstroke structural states (M* and M**) coexist in the S1.ADP.V(i) biochemical state. We propose that the pre-powerstroke state M** is characterized by two structural states that could reflect flexibility between the converter and N-terminal domains of myosin.
Nesmelov
Yuri E
YE
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota Medical School, Minneapolis, MN55455, USA. nesme004@umn.edu
Agafonov
Roman V
RV
Burr
Adam R
AR
Weber
Ralph T
RT
Thomas
David D
DD
D000818:Animals; D003198:Computer Simulation; D004548:Elasticity; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D008956:Models, Chemical; D008958:Models, Molecular; D020409:Molecular Motor Proteins / Q000737:chemistry* / Q000648:ultrastructure*; D015336:Molecular Probe Techniques; D009038:Motion; D018482:Muscle, Skeletal / Q000737:chemistry*; D009218:Myosins / Q000737:chemistry* / Q000648:ultrastructure*; D011487:Protein Conformation; D011817:Rabbits; D013314:Stress, Mechanical
D020409:Molecular Motor Proteins; D009218:Myosins