Protein-lipid interactions at membrane surfaces: a deuterium and phosphorus nuclear magnetic resonance study of the interaction between bovine rhodopsin and the bilayer head groups of dimyristoylphosphatidylcholine

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Abstract
Rhodopsin, isolated from bovine retinal rod outer segment disk membranes, has been reconstituted into bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine which was deuterated in the terminal methyl groups of the choline polar head group. By use of a mixed detergent system of cholate and octyl glucoside to solubilize the phospholipid and rhodopsin, 15 membrane complexes of predetermined phospholipid to rhodopsin mole ratios of between 350:1 and 65:1 have been produced by exhaustive dialysis and studied by a variety of techniques. Electron micrographs of replicas from freeze-fractured membrane complexes showed that the majority of the lipid, for all rhodopsin:phospholipid ratios, was contained in large bilayer vesicles with diameters in excess of 400 nm. Complexes produced with rhodopsin from frozen retina produced an absorption maximum at 478 nm after photobleaching whereas rhodopsin from fresh retina could be bleached more completely to an absorption maximum at 380 nm. Deuterium nuclear magnetic resonance (NMR) spectra from the lipid head groups of bilayers above the gel to liqud-crystalline phase transition temperature were shown to be sensitive in a systematic way to the presence of rhodopsin which could be bleached to 380 nm. The measured quadrupole splittings, taken as the separation of the turning points of the recorded NMR spectra, decreased from a value of 1.28 kHz for protein-free bilayers to approximately 0.40 kHz for bilayers containing 65 molecules of phospholipid for each rhodopsin at 32.degree. C. However, complexes formed by using rhodopsin from frozen retina or which had been fixed with glutaraldehyde displayed deuterium NMR spectra similar to protein-free phospholipid bilayers with no sensitivity to rhodopsin content and were thought to contain aggregated rhodopsin. Measurements of the total deuterium NMR spectral intensity recorded were compared with chemical and radiolabel determinations and showed that, within the error of the method, all the deuterated phospholipid was observed in the recorded spectra. No change in the high-frequency segmental motions of the lipid head groups was measured from deuterium NMR spin-lattice relaxation time measurements for the deuterated lipid groups. The phosphorus-31 NMR spectra of the lipid phosphate groups were typical for lipid bilayers at all rhodopsin:lipid ratios and were insensitive to the presence of rhodopsin, having a chemical shift anisotropy of approximately-45 ppm. For complexes which showed sensitivity to rhodopsin, the changes in the deuterium NMR spectra were shown, from control experiments, to be due only to the protein and not to residual detergents in the complexes. The NMR results suggest that nonaggregated rhodopsin in phopsholipid bilayers may cause relatively small and nonspecific perturbations of the polar choline head group. Fast exchange of the lipid between the protein surface and the bulk phase has also been demonstrated.

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