Orientation and rotational dynamics of spin-labeled phalloidin bound to actin in muscle fibers.
We have used electron paramagnetic resonance spectroscopy (EPR) to investigate the orientational distribution of actin in thin filaments of glycerinated muscle fibers in rigor, relaxation, and contraction. A spin-labeled derivative of a mushroom toxin, phalloidin (PHSL), was bound to actin in the muscle fibers (PHSL-fibers). The EPR spectrum of unoriented PHSL-labeled myofibrils consisted of three sharp lines with a splitting between the outer extrema (2T parallel') of 42.8 +/- 0.1 G, indicating that the spin labels undergo restricted nanosecond rotational motion within an estimated half-cone angle of 76 degrees. When the PHSL-fiber bundle was oriented parallel to the magnetic field, the splitting between the zero-crossing points (2T') was 42.7 +/- 0.1 G. When the fiber bundle was perpendicular to the magnetic field, 2T' decreased to 34.5 +/- 0.2 G. This anisotropy shows that the motion of the probe is restricted in orientation by its binding site on actin, so that the EPR spectrum of PHSL-fiber bundles would be sensitive to small changes in the mean axial orientation of the PHSL-actin interface. No differences in the EPR spectra were observed in fibers during rigor, relaxation, or contraction, indicating that the mean axial orientation of the PHSL binding site changes by less than 5 degrees, and that the amplitude of nanosecond probe rotational motion, which should be quite sensitive to the local environment of the phalloidin, changes by no more than 1 degree.(ABSTRACT TRUNCATED AT 250 WORDS)
Naber
N
N
Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0524.
Ostap
E M
EM
Thomas
D D
DD
Cooke
R
R
D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D004578:Electron Spin Resonance Spectroscopy; D009119:Muscle Contraction; D009126:Muscle Relaxation; D009132:Muscles / Q000187:drug effects / Q000378:metabolism*; D010590:Phalloidine / Q000378:metabolism* / Q000494:pharmacology; D011817:Rabbits; D013113:Spin Labels
D000199:Actins; D013113:Spin Labels; D010590:Phalloidine