We have investigated the molecular features of recombinant membranes that are necessary for the photochemical function of rhodopsin. The magnitude of the metarhodopsin I to metarhodopsin II phototransient following a 25% +/- 3% bleaching flash was used as a criterion of photochemical activity at 28 degrees C and pH 7.0. Nativelike activity of rhodopsin can be reconstituted with an extract of total lipids from rod outer segment membranes, demonstrating that the protein is minimally perturbed by the reconstitution protocol. Rhodopsin photochemical activity is enhanced by phosphatidylethanolamine head groups and docosahexaenoyl (22:6 omega 3) acyl chains. An equimolar mixture of phosphatidylethanolamine and phosphatidylcholine containing 50 mol% docosahexaenoyl chains results in optimal photochemical function. These results suggest the importance of both the head-group and acyl chain composition of the rod outer segment lipids in the visual process. The extracted rod lipids and those lipid mixtures favoring the conformational change from metarhodopsin I to II can undergo lamellar (L alpha) to inverted hexagonal (HII) phase transitions near physiological temperature. Interaction of rhodopsin with membrane lipids close to a L alpha to HII (or cubic) phase boundary may thus lead to properties which influence the energetics of conformational states of the protein linked to visual function.

To gain a better understanding of the biological roles of polyunsaturated phospholipids, deuterium (2H) NMR studies have been conducted of 1 -perdeuteriopalmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine, an asymmetric or mixed-chain saturated-polyunsaturated phospholipid, in the liquid crystalline (La) phase. The palmitoyl (16:O) chain at the glycerol sn-1 position was labeled with 2H by perdeuteration, whereas the polyunsaturated, docosahexaenoyl (22:6w3) chain at the sn-2 position was unlabeled, Le., protiated. The 2H NMR results were compared to studies of 1,2-diperdeuteriopalmitoyl-snglycero-3-phosphocholine, in which both the sn-1 and sn-2 palmitoyl chains were perdeuterated, as well as l-palmitoyl-2perdeuteriopalmitoyl-sn-glycero-3-phosphocholine, in which only the sn-2 chain was perdeuterated. Multilamellar phospholipid dispersions containing 50 wt % H 2 0 were employed, and 2H NMR spectra were obtained using quadrupolar echo methods at a magnetic field strength of 8.5 T. The experimental 2H NMR spectra were numerically deconvoluted (de-Paked) to yield subspectra corresponding to the parallel bilayer orientation with respect to the main applied magnetic field. The increased resolution of the de-Paked subspectra enabled profiles of the segmental order parameters of the individual C-2H bonds, denoted by lScD(i)1, to be derived as a function of chain position. Significant differences in the 2H N M R spectra and derived JScD!i)l profiles of the per-*H-16:0 chains of the polyunsaturated and saturated bilayers were found. Based on simplified statistical mechanical theories, the differences can be interpreted in terms of an increase in the configurational freedom of the palmitoyl chains in the polyunsaturated bilayer, relative to bilayers of phosphatidylcholines with two identical saturated chains. The increased configurational freedom may correspond to an increase in the equilibrium area per chain in the case of the polyunsaturated bilayer. Possible further interpretations of the results in terms of the thickness of the hydrocarbon region and the presence or lack of interdigitation of the polyunsaturated and saturated acyl chains are also briefly discussed. We conclude that the configurational properties of the acyl chains of polyunsaturated bilayers are significantly different from those of saturated phospholipid bilayers.

Polyunsaturated fatty acids are widely distributed components of biological membranes and are believed to be involved in many biological functions. However, the mechanisms by which they act on a molecular level are not understood. To further investigate the unique properties of omega 3 polyunsaturated phospholipid bilayers, deuterium nuclear magnetic resonance (2H NMR) studies have been made of the liquid-crystalline (L alpha) and gel phases of a homologous series of mixed-chain phosphatidylcholines containing docosahexaenoic acid: (per-2H-n:0)(22:6)PC, where n = 12, 14, 16, and 18. The moments of the 2H NMR lineshapes have been evaluated, and from these the warming and cooling main phase transition temperatures were determined. The transition temperatures of the mixed-chain series were found to be significantly lower than those of the corresponding lipids in the disaturated series, di(per-2H-n:0)PC, with hystereses ranging from 2 to 14 degrees C. Distinct effects of the docosahexaenoyl chain on bilayer order were found, though these effects varied across the mixed-chain series. In evaluating the moment data, an empirical method for normalizing the moments with respect to differences in temperature was applied, in addition to using the reduced temperature method. For the systems studied here, the method of normalization had no significant effect on the interpretation of the moment data.

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The characteristic 2H NMR signatures of the phospholipid (I) in the Lα phase, in which the palmitoyl chain is perdeuterated, are reported.