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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VDAC forms the major pathway for metabolites across the mitochondrial outer membrane. The regulation of the gating of VDAC channels is an effective way to control the flow of metabolites into and out of mitochondria. Here we present evidence that actin can modulate the gating process of Neurospora crassa VDAC reconstituted into membranes made with phosphatidylcholine. An actin concentration as low as 50 nM caused the VDAC-mediated membrane conductance to drop by as much as 85% at elevated membrane potentials. Actin's effect could be quickly reversed by adding pronase to digest the protein. alpha-Actin, from mammalian muscle, has a stronger effect than beta- and gamma-actin from human platelets. The monomeric form of actin, G-actin, is effective. Stabilization of the fibrous form, F-actin, with the mushroom toxin, phalloidin, blocks the effect of actin on VDAC, indicating that F-actin might be ineffective. Cytochalasin B did not interfere with the ability of actin to favor VDAC closure. DNase- did effectively block actin's effect on VDAC, and VDAC decreased actin's inhibitory effect on DNase-I activity, indicating that N. crassa VDAC competes with DNase-I for the same binding site on actin. The actin-VDAC interaction might be a mechanism by which actin regulates energy metabolism.

A Flory-Huggins-type lattice model of actin polymerization under equilibrium conditions is employed to analyze new spectroscopic measurements for the extent of actin polymerization ⌽ as a function of temperature T, salt concentration ͓KCl͔, and the initial concentration of actin monomers ͓G 0 ͔. The theory subsumes existing mechanisms for actin monomer initiation, dimerization, and chain propagation. The extent of polymerization ⌽ increases with T to an unanticipated maximum, and the calculations explain this unusual effect as arising from a competition between monomer activation, which diminishes upon heating, and propagating chain growth, which increases upon heating. The actin polymerization is described as a rounded phase transition, and the associated polymerization temperature T p depends strongly, but nearly linearly on ͓G 0 ͔ and ͓KCl͔ over the concentration regimes investigated. Our findings support the suggestion that physicochemical changes can complement regulatory proteins in controlling actin polymerization in living systems.

We have obtained high-field (11.7-T) proton and carbon-13 Fourier transform (FT) nuclear magnetic resonance (NMR) spectra of egg lecithin and egg lecithin-cholesterol (1:1) multibilayers, using "magic-angle" sample spinning (MASS) techniques, and sonicated egg lecithin and egg lecithin-cholesterol (1:1) vesicles, using conventional FT NMR methods. Resolution of the proton and carbon-13 MASS NMR spectra of the pure egg lecithin samples is essentially identical with that of sonicated samples, but spectra of the unsonicated lipid, using MASS, can be obtained very much faster than with the more dilute, sonicated systems. With the 1:1 lecithin-cholesterol systems, proton MASS NMR spectra are virtually identical with conventional FT spectra of sonicated samples, while with 13C NMR, we demonstrate that most 13C nuclei in the cholesterol moiety can be monitored, even though these same nuclei are essentially invisible, i.e., are severely broadened, in the corresponding sonicated systems. In addition, 13C MASS NMR, spectra can again be recorded much faster than with sonicated samples, due to concentration effects. Taken together, these results strongly suggest there will seldom be need in the future to resort to ultrasonic disruption of lipid bilayer membranes in order to obtain high-resolution proton or carbon-13 NMR spectra.

Studies of the dependence of actin polymerization on thermodynamic parameters are important for understanding processes in living systems, where actin polymerization and depolymerization are crucial to cell structure and movement. We report measurements of the extent of polymerization, ⌽, of rabbit muscle actin as a function of temperature ͓Tϭ(0-35)°C͔, initial G-actin concentration ͓͓G 0 ͔ϭ(1-3) mg/ml͔, and initiating salt concentration ͓͓KCl͔ϭ͑5-15͒ mmol/l with bound Ca 2ϩ ], in H 2 O and D 2 O buffers and in the presence of adenosine triphosphate ͑ATP͒. A preliminary account of the data and analysis for H 2 O buffers has appeared previously ͓P.