Regulation of membrane proteins by dietary lipids: effects of cholesterol and docosahexaenoic acid acyl chain-containing phospholipids on rhodopsin stability and function |
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Authors: | Bennett Michael P Mitchell Drake C |
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Affiliation: | Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland |
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Abstract: | Purified bovine rhodopsin was reconstituted into vesicles consisting of 1-stearoyl-2-oleoyl phosphatidylcholine or 1-stearoyl-2-docosahexaenoyl phosphatidylcholine with and without 30 mol % cholesterol. Rhodopsin stability was examined using differential scanning calorimetry (DSC). The thermal unfolding transition temperature (Tm) of rhodopsin was scan rate-dependent, demonstrating the presence of a rate-limited component of denaturation. The activation energy of this kinetically controlled process (Ea) was determined from DSC thermograms by four separate methods. Both Tm and Ea varied with bilayer composition. Cholesterol increased the Tm both the presence and absence of docosahexaenoic acid acyl chains (DHA). In contrast, cholesterol lowered Ea in the absence of DHA, but raised Ea in the presence of 20 mol % DHA-containing phospholipid. The relative acyl chain packing order was determined from measurements of diphenylhexatriene fluorescence anisotropy decay. The Tm for thermal unfolding was inversely related to acyl chain packing order. Rhodopsin kinetic stability (Ea) was reduced in highly ordered or disordered membranes. Maximal kinetic stability was found within the range of acyl chain order found in native bovine rod outer segment disk membranes. The results demonstrate that membrane composition has distinct effects on the thermal versus kinetic stabilities of membrane proteins, and suggests that a balance between membrane constituents with opposite effects on acyl chain packing, such as DHA and cholesterol, may be required for maximum protein stability. |
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