Regulatory and Catalytic Domain Dynamics of Smooth Muscle Myosin Filaments

Abstract

Domain dynamics of the chicken gizzard smooth muscle myosin catalytic domain (heavy chain Cys-717) and regulatory domain (regulatory light chain Cys-108) were determined in the absence of nucleotides using saturation-transfer electron paramagnetic resonance. In unphosphorylated synthetic filaments, the effective rotational correlation times, τ r , were 24 ± 6 μs and 441 ± 79 μs for the catalytic and regulatory domains, respectively. The corresponding amplitudes of motion were 42 ± 4° and 24 ± 9° as determined from steady-state phosphorescence anisotropy. These results suggest that the two domains have independent mobility due to a hinge between the two domains. Although a similar hinge was observed for skeletal myosin (Adhikari and Fajer (1997) Proc. Natl. Acad. Sci. U.S. A. 94, 9643-9647. Brown et al. (2001) Biochemistry 40,[8283][8284][8285][8286][8287][8288][8289][8290][8291], the latter displayed higher regulatory domain mobility, τ r = 40 ± 3 μs, suggesting a smooth muscle specific mechanism of constraining regulatory domain dynamics. In the myosin monomers the correlation times for both domains were the same (~4 μs) for both smooth and skeletal myosin, suggesting that the motional difference between the two isoforms in the filaments was not due to intrinsic variation of hinge stiffness. Heavy chain/regulatory light chain chimeras of smooth and skeletal myosin pinpointed the origin of the restriction to the heavy chain and established correlation between the regulatory domain dynamics with the ability of myosin to switch off but not to switch on the ATPase and the actin sliding velocity. Phosphorylation of smooth muscle myosin filaments caused a small increase in the amplitude of motion of the regulatory domain (from 24 ± 4° to 36 ± 7°) but did not significantly affect the rotational correlation time of the regulatory domain (441 to 408 μs) or the catalytic domain (24 to 17 μs). These data are not consistent with a stable interaction between the two catalytic domains in unphosphorylated smooth muscle myosin filaments in the absence of nucleotides. † This material is based upon work supported by the National Science Foundation under Grant No. 0346650, the AHA GIA 0455236B to P.G.F., and NIH AR40917 to C.R.C. and TCMRC 90103 to H.-C.L. L.S. is a recipient of American Heart Graduate Fellowship. *Author to whom correspondence should be addressed. Mailing address: Inst. Molecular Biophysics, Florida State University, Tallahassee, FL 32306. Tel: 850-645-1335. Fax: 850-644-1366. fajer@magnet.fsu.edu. ‡ Florida State University. § Tzu-Chi University. || These authors contributed equally. ⊥ University of Nevada School of Medicine. HHS Public Access Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptMyosins are a superfamily of motor proteins that convert the chemical energy derived from ATP hydrolysis to mechanical energy by generating movement or force. Myosin II, the major protein from muscle, consists of two globular head domains and a coiled-coil tail or rod domain. At physiological...

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