Rotational dynamics of actin-bound myosin heads in active myofibrils

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Abstract
We have used saturation-transfer electron paramagnetic resonance (ST-EPR) to measure the submillisecond rotational motions of actin-bound myosin heads in active myofibrils. The cross-bridges were spin-labeled with a maleimide nitroxide derivative (MSL) that has previously been shown to undergo microsecond rotational motions on actin-bound myosin heads in solution during steady-state ATPase activity at low ionic strength (Berger, C. L., Svensson, E. C., & Thomas, D. D. (1989) Proc. Nutl. Acud. Sci. U.S.A. 86,85731. To determine whether this is also true for cross-bridges in the myofibrillar lattice under physiological buffer conditions, we have performed ST-EPR experiments during the brief steady state following photolysis of caged ATP in a suspension of spin-labeled myofibrils. The myofibrils were partially cross-linked with EDC ( 1-ethyl-3-( 3-(dimethylamino)propyl)carbodiimide) to prevent their shortening upon activation. The fraction of actin-attached myosin heads was determined biochemically at physiological ionic strength in the active myofibrils, using the proteolytic rates acto-myosin binding assay (Duong, A. M., & Reisler, E. (1989) Biochemistry 28,35021. These data were then used to correct the ST-EPR spectra of active myofibrils for the presence of unattached myosin heads, which were assumed to undergo the same motions as in relaxation. At physiological ionic strength (p = 165 mM), actin-bound myosin heads were found to have considerable microsecond rotational motion (7, = 3.5 f 1.1 ps) in the active myofibrils. Similar results (7, = 3.2 f 0.8 ps) were obtained with active myofibrils at low ionic strength (p = 45 mM), confirming the work done in solution. Thus, under physiological conditions and even within the constraints of the myofibrillar lattice, actively cycling actin-attached myosin heads are rotationally mobile on the microsecond time scale. Since partially EDC-fixed myofibrils are an excellent analog of isometrically contracting muscle fibers in solution, it is likely that these microsecond rotational motions are directly related to the molecular mechanism of muscle contraction in vivo. Muscle contraction involves the cyclic interaction between actin and myosin, whereby the chemical free energy of adenosine 5'-triphosphate (ATP) hydrolysis is converted into mechanical work (Lymn & Taylor, 1971; Eisenberg & Hill, 1985). In particular, force generation and myofilament sliding are thought to occur via conformational changes in the myosin head (subfragment 1, Sl), while bound to actin during the acto-myosin ATPase cycle (Huxley, 1969; Huxley & Sim- mons, 1971; Huxley & Kress, 1985). Therefore, direct measurements of Sl's structural dynamics during the con- tractile process are imperative for understanding the molecular mechanism of muscle contraction. Spectroscopic techniques such as electron paramagnetic resonance (EPR) and time- resolved phosphorescence anisotropy (TPA) provide excellent means by which to monitor protein motions at specific sites in macromolecular complexes such as acto-myosin (reviewed by Thomas (1 987)). ~ ~~ ~~ __~ ~~~ ___~~~~

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