High-field proton magic-angle sample-spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy is shown to yield high-resolution 'H spectra of smectic, nematic and hexagonal-I1 phase lipids, from which isotropic chemical shifts, order parameters and relaxation times (q, qp and T,) can be determined. Such experiments are possible because of the special form of the dipolar Hamiltonian in such systems. Resolution is about the same as that obtained with sonicated systems, using conventional NMR techniques. We also show that 13C MAS NMR spectra, of both fluid and solid phases, are even better resolved, and in some cases resonances can be observed in MAS NMR spectra which are not observable in sonicated systems. For example, essentially all of the carbon atoms in cholesterol (CHOL) can be readily detected and assigned in a lecithin-CHOL bilayer, using MAS, while few can be seen in sonicated bilayers. This leads directly to the observation of cholesterol in intact biological membranes, such as human myelin, where over 50 peaks can be observed, and ca. 40 of these resonances can be assigned to specific, single-carbon-atom sites in the membrane. In addition, a number of experiments with massively deuterated lipids are reported. Combination of cross-polarization techniques with MAS, and difference spectroscopy, leads to the observation of essentially pure sterol spectra (in the presence of lipid) and pure lipid spectra (in the presence of CHOL). Analysis of chemical-shift results indicates a substantial deshielding of chain carbon atom resonances caused by the presence of CHOL, due presumably to increased trans chain segments, an effect mirrored in variable temperature spectra of human myelin, and in goldfish myelin. Taken together, these results suggest a resurgence in NMR studies of membranes may soon occur.

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The nanomechanical properties of the coiled-coils of myosin are fundamentally important in understanding muscle assembly and contraction. Force spectra of single molecules of double-headed myosin, single-headed myosin, and coiled-coil tail fragments were acquired with an atomic force microscope and displayed characteristic triphasic force-distance responses to stretch: a rise phase (R) and a plateau phase (P) and an exponential phase (E). The R and P phases arise mainly from the stretching of the coiled-coils, with the hinge region being the main contributor to the rise phase at low force. Only the E phase was analyzable by the worm-like chain model of polymer elasticity. Restrained molecular mechanics simulations on an existing x-ray structure of scallop S2 yielded force spectra with either two or three phases, depending on the mode of stretch. It revealed that coiled-coil chains separate completely near the end of the P phase and the stretching of the unfolded chains gives rise to the E phase. Extensive conformational searching yielded a P phase force near 40 pN that agreed well with the experimental value. We suggest that the flexible and elastic S2 region, particularly the hinge region, may undergo force-induced unfolding and extend reversibly during actomyosin powerstroke.

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