Backbone and side-chain motion in myosin, subfragment 1, and rod determined by natural abundance carbon-13 NMR.
We report natural abundance carbon-13 nuclear magnetic resonance studies of myosin, subfragment 1 (S-1), and myosin rod in solution and of myosin filaments. We measured solution spectra at 37.7 MHz, 20 degrees C with scalar proton decoupling. If the native proteins were rigid particles, only S-1 would have observable intensity. In fact, 30% of myosin, 70% of S-1, and 30% of rod carbons are observable. Observable carbons possess rotational correlation times less than 10(-7) s which effectively average 13C-1H dipolar coupling and anisotropic chemical shift interactions. Short alpha-carbon region spin-lattice relaxation times (T1) and nonminimal 13C-1H nuclear Overhauser enhancements suggest restricted nanosecond motions of backbone atoms. This is direct evidence for internal motion of backbone and side-chain carbons in myosin and its fragments. Double resonance experiments at 15.1 MHz and 5 degrees C with pelleted myosin filaments detected carbon atoms in rigid domains. While most (80%) aliphatic carbons are strongly 13C-1H dipolar coupled due to limited motion, they have short T1 values and large nuclear Overhauser enhancement values; this is evidence for high frequency restricted motion. Cross-polarization experiments show that the 13C carbonyl line width is motionally narrowed, suggesting broad backbone motions in the 100 mus range. Thus, motion in filaments is highly anisotropic.