The objectives of this project are to: (1) study structure and dynamics of membranes composed of lipids containing polyunsaturated fatty acids such as docosahexaenoic acid (DHA) 22:6n-3, (2) study lipid-protein interactions related to lipid polyunsaturation and alcoholism, and (3) investigate the interaction of alcohol with proteins and lipids in biological membranes. (1) The membranes of brain synaptosomes and retinal rod outer segments contain 30-50 mol% of the six-fold unsaturated docosahexaenoic acid (DHA) as lipid hydrocarbon chains. One possible role of DHA is to alter membrane mechanical properties important for activity of receptor proteins. Using a magic angle spinning NMR experiment which re-couples 13C-1H dipolar interactions, assigned DHA order parameters were obtained. A unique membrane probe - perdeuterated DHA - was synthesized and incorporated into the lipid matrix. Twelve distinct order parameters were measured. Furthermore, the dimensions of the DHA chain unit cell were determined by x-ray diffraction. Order parameters of all methylene segments between double bonds in the hydrocarbon chain, and the order of the majority of double bonds is very low. Only the two methylene segments near the carboxyl group of DHA have order parameters that are comparable to values of more saturated chains. The low order is a reflection of both a change in bond geometry and an increase in chain motions. Experimental results were combined with results of simulations. The analysis suggests that DHA chains in membranes can exchange between looped, tilted, and extended conformations in rapid succession, providing increased flexibility to receptor-rich neural membranes. We developed quantitative methods for interpretation of NMR NOESY cross-relaxation rates between lipid resonances. In addition to providing information on lipid structure, these rates are sensitive to the dynamics of membrane reorganization in the correlation time range form pico- to microseconds. The comparison of experimental rates and rates from molecular dynamics simulations suggests that distance variation between protons caused by lateral diffusion of lipid molecules is the primary mechanism of cross-relaxation in lipids. The analysis quantifies the high degree of molecular disorder in biological membranes, showing a finite probability of close approach between even the most distant segments of neighboring lipid molecules (e.g. the methyl groups in the choline headgroup and the terminal methyl groups of the fatty acid chains). Intermolecular cross-relaxation rates are an ideal tool to study lateral lipid organization in the liquid-crystalline phase of lipids. Inhomogeneous lipid distribution and preferences in the interaction of lipid species, as well as preferences in the location of substances that incorporate into membranes can be detected. We developed approaches to conduct experiments on membrane samples oriented at solid interfaces and in lipid mixtures that orient spontaneously in the strong magnetic field of NMR instruments. The analysis of NMR lineshapes revealed the variable degree of mosaic spread in bilayer orientation for the different membranes. (2) There is evidence that a high content of DHA in retinal membranes modulates physical properties of membranes, creating an environment that is optimal for function of rhodopsin, the primary visual receptor, and a member of the G-protein coupled receptor family. We investigated this hypothesis by solid-state NMR methods. Rhodopsin was reconstituted into fully hydrated, solid-supported oriented multi-bilayer samples. Using 2H-labeled lipids, we compared lipid order parameters in the absence, and in the presence of the protein. We obtained highly resolved spectra from deuterated acyl chains in membranes containing a reconstituted integral membrane protein under physiological conditions. Oriented samples also improve NMR sensitivity enabling work with milligram-size samples. We have studied the phase diagram of the polyunsaturated 18:0-22:6 PE and demonstrated that it forms inverse hexagonal phases at all temperatures above the gel-fluid transition. The results suggest the existence of polyunsaturated lipid-induced membrane curvature stress that is likely to modulate the degree of activation of membrane incorporated receptors like rhodopsin. (3) Ethanol can act at multiple sites, with variable emphasis on interaction via the lipid matrix or via direct interaction with the protein, depending on the specific protein system involved. We propose that the binding of ethanol molecules to the lipid matrix of biomembranes is an important event in the action of ethanol on biological matter. We studied the interaction of ethanol with saturated, mono-, and polyunsaturated membranes quantitatively by MAS NOESY NMR. The resolution of resonance lines allows detection of 13-16 proton signals from lipid, ethanol, and water. Results of NMR measurements were combined with atomic-level molecular dynamics simulations to provide a deeper interpretation of experimental results. The site of ethanol interaction with the lipids is the primary factor that determines NMR cross-relaxation rates. Differences in correlation times and motional amplitudes of lipid segments play a secondary role. In particular, magnetization transfer to the headgroup choline resonance was somewhat lower than expected, due to fast lipid dynamics. As observed previously for lipid-lipid cross-relaxation, the rates scale with translational diffusion rates of ethanol in the bilayer. Ethanol resides mostly in the water phase, but binds for brief periods of time, of the order of nanoseconds, to the polar groups of the lipid/water interface, primarily to lipid phosphate groups. This temporary interaction introduces anisotropy into the motion of ethanol, with ethanol methylene C-D order parameters of 0.06. Cross-relaxation is strongest between ethanol and lipid resonances from the lipid/water interface including the glycerol, upper hydrocarbon chain, and lipid headgroup regions. There is evidence that the ethanol molecule in membranes is oriented preferentially with its methyl group toward the hydrophobic bilayer core. Cross-relaxation between ethanol and lipid hydrocarbon chains methyl is a reflection of both lipid hydrocarbon chain upturns and brief excursions of ethanol molecules into the upper region of lipid hydrocarbon chains. Overall, the probability of ethanol penetration into the center of the hydrophobic core of membranes is extremely low.
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