LIPID HEADGROUP AND ACYL CHAIN COMPOSITION MODULATE THE MI-MII EQUILIBRIUM OF RHODOPSIN IN RECOMBINANT MEMBRANES
LIPID HEADGROUP AND ACYL CHAIN COMPOSITION MODULATE THE MI-MII EQUILIBRIUM OF RHODOPSIN IN RECOMBINANT MEMBRANES
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DOI：
10.1021/bi00060a040
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发表时间：
1993-03-09
期刊：
影响因子：
2.9
通讯作者：
BROWN, MF
中科院分区：
文献类型：
--
作者：
GIBSON, NJ;BROWN, MF
目前视觉功能的范式集中在变视紫红质 I (MI) 到变视紫红质 II (MII) 构象转变上，作为细胞内酶级联的触发因素，导致视网膜杆的兴奋。我们使用闪光光解技术研究了膜脂成分对视觉信号转导中这一关键触发事件的影响。将牛视紫红质与各种磷脂结合形成膜重组体，其中脂质酰基链组成与卵磷脂酰胆碱 (PC) 保持恒定，而脂质头基的特性发生变化。研究了在 28 摄氏度下通过光化闪光在这些重组体中产生的 MII/MI 比率作为 pH 的函数。将结果与天然视网膜杆外节 (ROS) 膜、总天然 ROS 脂质重组体和二肉豆蔻酰磷脂酰胆碱 (DMPC) 重组体中观察到的视紫红质的光化学功能进行比较。在掺入来自卵 PC 的脂质的膜重组体中，以及在总 ROS 脂质对照和天然 ROS 盘膜中，发现 MI 和 MII 在毫秒时间尺度上以 pH 依赖性酸碱平衡共存。视紫红质与卵 PC 的重组体，无论是单独使用还是与卵 PC 衍生的磷脂酰乙醇胺 (PE) 或磷脂酰丝氨酸 (PS) 组合，在 pH 7.0 下表现出显着降低的光化学活性。然而，所有包含具有不饱和酰基链的磷脂的重组体都能够在 pH 5.0 下完全产生类似天然的 MII，这证实了先前的结果 [Gibson, N. J., & Brown, M. F. (1990) Biochem.生物物理学。资源。交流。 169、1028-1034]。由此可见，鸡蛋 PC 衍生的酰基链对 MI 和 MII 态施加的能量限制可以通过 H+ 离子活性的增加来抵消。数据表明，膜脂成分的主要作用是改变视紫红质 MI-MII 构象平衡的表观 pK [Gibson, N. J., & Brown, M. F. (1991) Biochem.生物物理学。资源。交流。 176、915-921]。仅含有磷酸胆碱头基的重组体表现出最低的表观 pK 值，而 50 mol% PE 或 15 mol% PS 的存在则增加了表观 pK。在具有卵 PC 衍生链和天然样头基组成的重组体中无法获得完全天然样功能，表明天然视网膜杆膜脂质的多不饱和二十二碳六烯酸 (DHA) 链 (22:6omega3) 的重要作用。 MI-MII 转变的温度研究使得能够研究脂质对与功能直接相关的膜蛋白构象变化的热力学参数的影响。热力学状态变量的变化表明视紫红质可能在 MII 状态下部分展开，导致信号转导 G 蛋白的识别位点暴露。最后，根据膜脂双层的性质讨论了结果，包括双层静电的影响以及与蛋白质/脂质和脂质/水界面相关的散装材料性质。由于涉及脂质/水界面的横向和/或曲率应力而产生的相对较小的变化足以解释重组体中 MI-MII 转变的自由能变化。PE 头基与天然视网膜杆脂质中庞大的 DHA 链的组合促进了非层状相的形成；一种可能性是涉及膜-脂质/水界面的曲率自由能。这些发现表明膜脂组成直接影响视紫红质的光化学活性，从而暗示视觉过程分子机制中膜脂双层的特性。
A current paradigm for visual function centers on the metarhodopsin I (MI) to metarhodopsin II (MII) conformational transition as the trigger for an intracellular enzyme cascade leading to excitation of the retinal rod. We investigated the influences of the membrane lipid composition on this key triggering event in visual signal transduction using flash photolysis techniques. Bovine rhodopsin was combined with various phospholipids to form membrane recombinants in which the lipid acyl chain composition was held constant at that of egg phosphatidylcholine (PC), while the identity of the lipid headgroups was varied. The ratio of MII/MI produced in these recombinants by an actinic flash at 28-degrees-C was studied as a function of pH. The results were compared to the photochemical function observed for rhodopsin in native retinal rod outer segment (ROS) membranes, in total native ROS lipid recombinants, and in dimyristoylphosphatidylcholine (DMPC) recombinants. In membrane recombinants incorporating lipids derived from egg PC, as well as in the total ROS lipids control and the native ROS disk membranes, MI and MII were found to coexist in a pH-dependent, acid-base equilibrium on the millisecond time scale. The recombinants of rhodopsin with egg PC, either alone or in combination with egg PC-derived phosphatidylethanolamine (PE) or phosphatidylserine (PS), exhibited substantially reduced photochemical activity at pH 7.0. However, all recombinants comprising phospholipids with unsaturated acyl chains were capable of full native-like MII production at pH 5.0, confirming previous results [Gibson, N. J., & Brown, M. F. (1990) Biochem. Biophys. Res. Commun. 169, 1028-1034]. It follows that energetic constraints on the MI and MII states imposed by egg PC-derived acyl chains can be offset by increased activity of H+ ions. The data reveal that the major effect of the membrane lipid composition is to alter the apparent pK for the MI-MII conformational equilibrium of rhodopsin [Gibson, N. J., & Brown, M. F. (1991) Biochem. Biophys. Res. Commun. 176, 915-921]. Recombinants containing only phosphocholine headgroups exhibited the lowest apparent pK values, whereas the presence of either 50 mol % PE or 15 mol % PS increased the apparent pK. The inability to obtain full native-like function in recombinants having egg PC-derived chains and a native-like headgroup composition indicates a significant role of the polyunsaturated docosahexaenoic acid (DHA) chains (22:6omega3) of the native retinal rod membrane lipids. Temperature studies of the MI-MII transition enabled an investigation of lipid influences on the thermodynamic parameters of a membrane protein conformational change linked directly to function. The changes in thermodynamic state variables suggest that rhodopsin may be partially unfolded in the MII state, leading to exposure of recognition sites for the signal transducing G protein. Finally, the results are discussed in terms of properties of the membrane lipid bilayer, including the influences of bilayer electrostatics as well as bulk material properties associated with the protein/lipid and lipid/water interfaces. Relatively small changes due to lateral and/or curvature stresses involving the lipid/water interface are sufficient to explain the free energy shifts for the MI-MII transition among the recombinants.The combination of PE headgroups together with bulky DHA chains in the native retinal rod lipids promotes formation of nonlamellar phases; one possibility is that the curvature free energy of the membrane-lipid/water interface is involved. These findings indicate that the membrane lipid composition influences directly the photochemical activity of rhodopsin, thereby implicating properties of the membrane lipid bilayer in the molecular mechanism of the visual process.