The quantitative measurement of rotational motion of the subfragment‐1 region of myosin by saturation transfer EPR spectroscopy

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Abstract
According to current models of muscle contraction (Huxley, H. E., Science 164: 1356–1366 [19691]), motion of flexible myosin crossbridges is essential t o the contractile cycle. Using a spin-label analog of iodoacetamide bound to the subfragment # 1 (S1) region of myosin, we have obtained rotational correlation times (τ2) for this region of the molecule with the ultimate goal of making quantitative measurements of the motion of the crossbridges under conditions comparable to those in living, contracting muscle. We used the newly developed technique of saturation transfer electron paramagnetic resonance spectroscopy (Hyde, J.S., and Thomas, D.D., Ann. N.Y. Acad. Sci. 222:680–692 [1973]), which is uniquely sensitive t o rotational motion in the range of 10−7–10−3 sec. Our results indicate that the spin label is rigidly bound to S1 (τ2 for isolated S1 is 2 × 10−7 sec) and that the motion of the label reflects the motion of the S1 region of myosin. The value of τ2 for the S1 segment of myosin is less than twice that for isolated S1, while the molecular weights differ by a factor of 4, indicating flexibility of myosin in agreement with the conclusions of Mendelson et aL (Biochemistry 12:2250–2255 [1973]). Adding F-actin increases τ2 in either myosin or isolated S1 by a factor of nearly 103, indicating rigid immobilization of S1 by actin. Formation of myosin filaments (at an ionic strength of 0.15 or less) increases τ2 by a factor of 10–30, depending o n the ionic strength, indicating a decrease of the rotational mobility of S1 in these aggregates. The remaining motion is at least a factor of 10 faster than would be expected for the filament itself, suggesting motion of the S1 region independent of the filament backbone but slower than in a single molecule. F-actin has a strong immobilizing effect on labeled S l in myosin filaments (in 0.137 M KC1), but the immobilization is less complete than that observed when F-actin is added t o labeled myosin monomers (in 0.5 M KC1). A spin-label analog of maleimide, attached to the SH-2 thiol groups of S1, is immobilized to a much lesser extent by F-actin than is the label on SH-1 groups. The maleimide label also was attached directly to F-actin and was sufficiently immobilized to suggest rigid binding to actin.