Regulation of membrane proteins by dietary lipids: Effects of cholesterol and docosahexaenoic acid acyl chain-containing phospholipids on rhodopsin stability and function
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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DOI：
10.1529/biophysj.107.122788
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发表时间：
2008-08-01
影响因子：
3.4
通讯作者：
Mitchell, Drake C.
中科院分区：
文献类型：
--
作者：
Bennett, Michael P.;Mitchell, Drake C.
将纯化的牛视紫红质重构为由 1-硬脂酰-2-油酰磷脂酰胆碱或 1-硬脂酰-2-二十二碳六烯酰磷脂酰胆碱（含或不含 30 mol% 胆固醇）组成的囊泡。使用差示扫描量热法（DSC）检查视紫质稳定性。视紫红质的热解折叠转变温度 (T-m) 与扫描速率相关，表明存在速率限制的变性成分。该动力学控制过程的活化能 (E-a) 通过四种不同的方法根据 DSC 热分析图确定。 T-m 和 Ea 均随双层组成而变化。无论是否存在二十二碳六烯酸酰基链 (DHA)，胆固醇都会增加 T-m。相反，在不存在 DHA 的情况下，胆固醇会降低 Ea，但在存在 20 mol% 含 DHA 的磷脂的情况下，胆固醇会升高 Ea。相对酰基链堆积顺序由二苯基己三烯荧光各向异性衰减的测量确定。热解折叠的 Tm 与酰基链堆积顺序成反比。高度有序或无序的膜中视紫红质动力学稳定性 (E-a) 降低。在天然牛杆外节圆盘膜中发现的酰基链序范围内发现了最大的动力学稳定性。结果表明，膜成分对膜蛋白的热稳定性和动力学稳定性具有明显的影响，并表明对酰基链堆积具有相反影响的膜成分（例如 DHA 和胆固醇）之间的平衡可能是最大蛋白质稳定性所必需的。
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 (T-m) of rhodopsin was scan rate-dependent, demonstrating the presence of a rate-limited component of denaturation. The activation energy of this kinetically controlled process (E-a) was determined from DSC thermograms by four separate methods. Both T-m and Ea varied with bilayer composition. Cholesterol increased the T-m 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 (E-a) 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.