Fluorescent fatty acid labels have been incorporated into the palmitoylation sites of rhodopsin and used to probe the membrane accessibility and location of these sites. The fluorescence properties of anthroyloxy and pyrenyl fatty acids bound to rhodopsin were investigated in a reconstituted vesicle system. Collisional quenching of fluorescence by stearic acid (DSA) labeled with doxyls in the 16, 12, and 5 positions was used to determine the membrane accessibility and disposition of the modifying fatty acids. To properly determine the membrane concentration of these quenchers, the dependence of the Stern-Volmer parameters on both quencher and vesicle concentration was determined. An analysis of these dependences provided a correction for partitioning of the quencher between the aqueous phase and the membrane. After this correction, the relative effectiveness of doxyl quenchers was 16-DSA > 12-DSA > 5-DSA. Parallel studies on free anthroyloxy and pyrenyl fatty acids incorporated into the reconstituted system showed the same dependence on quencher position. These results indicate that the labels at the palmitoylation sites of rhodopsin are situated in the membrane much as a free fatty acid. This anchoring of the palmitates in the membrane results in the formation of a fourth cytoplasmic loop.

In an effort to determine the functionality of palmitoylation in rhodopsin, a number of physicochemical properties of depalmitoylated rhodopsin were monitored. Approximately 70% of the rhodopsin was depalmitoylated in rod outer segments by a mild hydroxylamine treatment that resulted in minimal bleaching of rhodopsin. Subsequent purification by affinity chromatography could be used to remove hydroxylamine-bleached rhodopsin. Parallel physical studies were performed on both purified, detergent-solubilized rhodopsin and rhodopsin in rod outer segments. No effect was seen on the rate of metarhodopsin II formation for depalmitoylated rhodopsin. A small effect was seen in the biphasic behavior of the rate of retinal regeneration. The circular dichroism spectrum of depalmitoylated, purified rhodopsin was virtually identical to that of the native protein. These results suggest that depalmitoylation does not greatly affect the conformational structure of rhodopsin. Circular dichroism at 222 nm was used to monitor the thermal denaturation of depalmitoylated and native rhodopsin. A small but significant decrease in the in rod outer segments. In both cases, the van't Hoff parameters showed an increase in positive enthalpy for denaturation relative to the native state. This is largely counterbalanced by an increase in positive entropy relative to the native states. The circular dichroism of the "denatured" state showed a high alpha-helix content. Depalmitoylated rhodopsin had a lower helix content than native protein in this high-temperature state. The changes in the thermodynamics upon depalmitoylation were attributed to structural changes in the denatured state.

Two tandem cysteine residues in the carboxyl-terminal region of rhodopsin have been shown to be covalently linked to palmitate via thioester bonds (Ovchinnikov, Y. A., et al. (1988) FEBS Lett. 230, 1-5). We have synthesized a fluorescent analogue of palmitoyl coenzyme A (16-(9-anthroyloxy)hexadecanoyl coenzyme A ester) and incorporated the fluorescent derivative of palmitate into the protein in high yield (> 40%) through pretreatment of bovine rod outer segments with 1 M hydroxylamine and subsequent incubation with the fluorescent label. Covalent incorporation of label into protein was demonstrated by SDS-polyacrylamide gel electrophoresis. Proteolytic digestion of labeled rhodopsin in the disc membrane with papain and thermolysin verified the C-terminal location of the label. Treatment of SDS-solubilized, labeled rod outer segments with 10% beta-mercaptoethanol provided evidence that partial depalmitoylation may induce the formation of rhodopsin aggregates. Labeled, unbleached rhodopsin was purified by chromatography over hydroxyapatite and concanavalin A-agarose and reconstituted into dimyristoylphosphatidylcholine vesicles. SDS gels of the rhodopsin vesicle preparation verified that all unbound fluorescent label had been removed and that the thioester bond linking probe to protein was not labile.

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