Method for the determination of myosin head orientation from EPR spectra.
The determination of the iodoacetamide spin label orientation in myosin heads (Fajer, 1994) allows us for the first time to determine directly protein orientation from EPR spectra. Computational simulations have been used to determine the sensitivity of EPR to both torsional and tilting motions of myosin heads. For rigor heads (no nucleotide), we can detect 0.2 degree changes in the tilt angle and 4 degrees in the torsion of the head. Sensitivity decreases with increasing head disorder, but even in the presence of +/- 30 degrees disorder as expected for detached heads, 10 degree changes in the center of the orientational distribution can be detected. We have combined these numerical simulations with a Simplex optimization to compare the orientation of intrinsic heads, with the orientation of labeled extrinsic heads that have been infused into unlabeled muscle fibers. The near identity (within 2 degrees) of the orientational distribution in the two instances can be attributed to myosin elasticity taking up the mechanical strain induced by the mismatch of myosin and actin filament periodicity. A similar analysis of the spectra of fibers with ADP bound to myosin revealed a small (approximately 5 degrees-10 degrees) torsional reorientation, without a substantial change of the tilt angle (< 2 degrees).
Fajer
P G
PG
Institute of Molecular Biophysics, Florida State University, Tallahassee 32300.
D000199:Actins / Q000737:chemistry / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D003497:Cyclic N-Oxides; D003903:Deuterium; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D009119:Muscle Contraction; D009132:Muscles / Q000502:physiology; D015879:Myosin Subfragments / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D013113:Spin Labels
D000199:Actins; D003497:Cyclic N-Oxides; D015879:Myosin Subfragments; D013113:Spin Labels; C003028:N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide; D000244:Adenosine Diphosphate; D003903:Deuterium