The objectives of this project are to: (1) study structure and dynamics of membranes composed of lipids with 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) Insufficient supply to the developing brain of docosahexaenoic acid (22:6n3, DHA), or its omega-3 fatty acid precursors, results in replacement of DHA with docosapentaenoic acid (22:5n6, DPA), an omega-6 fatty acid that is lacking a double bond near the chain's methyl end. We investigated membranes of 1-stearoyl(d35)-2-docosahexaenoyl-sn-glycero-3-phosphatidylcholine and 1-stearoyl(d35)-2-docosapentaenoyl-sn-glycero-3-phosphatidylcholine to determine if the loss of this double bond alters membrane physical properties. Magic angle spinning NMR enabled almost liquid-like resolution of 1H- and 13C-resonances. We assigned resonances, and measured 2H order parameters, 1H-1H cross-relaxation rates, 1H-13C cross-polarization rates, and 13C spin-lattice relaxation rates. X-ray diffraction experiments yielded electron density profiles. Finally, we conducted molecular dynamics simulations of the membranes to aid interpretation of experimental results. Both polyunsaturated chains are motionally disordered to the point that intermolecular choline head group-chain methyl group contacts occur with low, but measurable, probability. The correlation times of polyunsaturated chain motions decrease in steps from double bond to double bond. The chain motions near the carbonyl group have significant contributions from correlation times in the nanosecond range while the motions near the terminal methyl group are dominated by correlation times of picoseconds. There are important differences between DHA- and DPA-containing lipids: the DHA chain with one additional double bond is more flexible at the methyl end and moves with shorter correlation times. Furthermore, the stearic acid paired with the DHA in mixed-chain lipids has significantly lower order parameters from the center of chains down to the terminal methyl group, indicating differences in the packing in the center of the bilayer. These differences are reflected in both the measured and calculated electron density profiles. Polyunsaturated chains have higher density near the lipid water interface while density of saturated chains is higher in the bilayer center. This asymmetry in distribution of chain densities is smaller for lipids with DPA compared to DHA. We propose that the function of integral membrane proteins like rhodopsin is altered by such differences in membrane structure and dynamics. (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 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. The interaction of ethanol with palmitoyloleoylphosphatidylcholine (POPC) phospholipid bilayers has been studied using a combination of nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) computer simulation. Measurements of nuclear Overhauser enhancement spectroscopy (NOESY) cross-relaxation rates were combined with atomic-level simulations to provide a highly detailed picture of the system. Ethanol resides in the water phase and interfacial regions, binding to the polar groups of the lipid/water interface, primarily to lipid phosphate groups, for brief periods of time on the order of nanoseconds. This temporary association 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 at the lipid/water interface, e.g. the glycerol and upper hydrocarbon chain regions. The MD simulations enabled us to calculate autocorrelation functions for proton dipole-dipole interactions, and, consequently, relaxation times and cross-relaxation rates. These analyses allow the measured cross-relaxation rates to be interpreted in terms of relative interaction strengths with the various segments of the lipid molecule. Cross-relaxation rates between ethanol and specific lipid resonances are primarily determined by the sites of interaction, with some modulation due to local differences in intramolecular lipid dynamics. As found previously for lipid-lipid cross-relaxation, the rates appear to scale with translational diffusion rates of ethanol. Thus, lower diffusion rates, as achieved by lowering temperature and/or water content of samples, result in higher cross-relaxation rates. The probability of finding ethanol in the bilayer center is extremely low. The low cross-relaxation rates between terminal methyl protons of hydrocarbon chains and ethanol are as much the result of infrequent chain upturns as of brief excursions of ethanol into the region of lipid hydrocarbon chains.
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