Effects of depalmitoylation on physicochemical properties of rhodopsin.
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.
Traxler
K W
KW
Department of Chemistry, University of Denver, Colorado 80208.
Dewey
T G
TG
D000818:Animals; D002417:Cattle; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002942:Circular Dichroism; D006358:Hot Temperature; D007700:Kinetics; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism*; D011489:Protein Denaturation; D012160:Retina / Q000378:metabolism; D012243:Rhodopsin / Q000031:analogs & derivatives / Q000737:chemistry* / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000737:chemistry; D013816:Thermodynamics
D010169:Palmitic Acids; D019308:Palmitic Acid; C013110:metarhodopsins; D012243:Rhodopsin