Slow Phospholipid Exchange between a Detergent-Solubilized Membrane Protein and Lipid-Detergent Mixed Micelles: Brominated Phospholipids as Tools to Follow Its Kinetics.
Slow Phospholipid Exchange between a Detergent-Solubilized Membrane Protein and Lipid-Detergent Mixed Micelles: Brominated Phospholipids as Tools to Follow Its Kinetics.
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慢慢的磷脂交换在洗涤剂 - 溶解的膜蛋白和脂质含量混合胶束之间：溴化磷脂作为遵循其动力学的工具。
DOI：
10.1371/journal.pone.0170481
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Lenoir G
中科院分区：
文献类型：
--
作者：
Montigny C;Dieudonné T;Orlowski S;Vázquez-Ibar JL;Gauron C;Georgin D;Lund S;le Maire M;Møller JV;Champeil P;Lenoir G
膜蛋白的功能在很大程度上依赖于其直接环境中存在的磷脂，当它们被去垢剂溶解以供进一步研究时，残留的磷脂也很关键。在这里，溴化磷脂酰胆碱（一种表现为不饱和磷脂酰胆碱的磷脂）被用来揭示磷脂交换或从洗涤剂混合胶束转移到洗涤剂溶解的膜蛋白（典型的 P 型 ATP 酶 SERCA1a）环境的动力学，其中色氨酸残基可以通过其直接环境中磷脂烷基链上存在的溴原子经历荧光猝灭。使用十二烷基麦芽糖苷作为去垢剂，发现（溴化）磷脂的交换比相同条件下去垢剂的交换慢得多，并且也比膜溶解慢得多，后者通过光散射变化得到证明。这种交换的动力学强烈依赖于温度。它还取决于混合胶束的总浓度，揭示了去污剂胶束与去污剂溶解的蛋白质碰撞的这种交换的主要作用。如果添加无脂 DDM 胶束而不是混合胶束来触发溴化磷脂酰胆碱从溶解的蛋白质解离，或者在交换过程中额外存在 C12E8 去污剂，则溴化磷脂从溶解的蛋白质向去污剂胶束的反向转移要快得多，这也强调了胶束/蛋白质界面的化学性质的作用。该使用溴化脂质的方案似乎对于揭示磷脂与去污剂溶解的膜蛋白之间的转移可能缓慢的动力学很有价值。独立地，在某些情况下，也可以使用连续记录蛋白质的活性，将活性的变化与特定磷脂的交换相关联，如使用 Drs2p/Cdc50p 复合物（一种具有脂质特异性结合位点的脂质翻转酶）所示。
Membrane proteins are largely dependent for their function on the phospholipids present in their immediate environment, and when they are solubilized by detergent for further study, residual phospholipids are critical, too. Here, brominated phosphatidylcholine, a phospholipid which behaves as an unsaturated phosphatidylcholine, was used to reveal the kinetics of phospholipid exchange or transfer from detergent mixed micelles to the environment of a detergent-solubilized membrane protein, the paradigmatic P-type ATPase SERCA1a, in which Trp residues can experience fluorescence quenching by bromine atoms present on phospholipid alkyl chains in their immediate environment. Using dodecylmaltoside as the detergent, exchange of (brominated) phospholipid was found to be much slower than exchange of detergent under the same conditions, and also much slower than membrane solubilization, the latter being evidenced by light scattering changes. The kinetics of this exchange was strongly dependent on temperature. It was also dependent on the total concentration of the mixed micelles, revealing the major role for such exchange of the collision of detergent micelles with the detergent-solubilized protein. Back-transfer of the brominated phospholipid from the solubilized protein to the detergent micelle was much faster if lipid-free DDM micelles instead of mixed micelles were added for triggering dissociation of brominated phosphatidylcholine from the solubilized protein, or in the additional presence of C12E8 detergent during exchange, also emphasizing the role of the chemical nature of the micelle/protein interface. This protocol using brominated lipids appears to be valuable for revealing the possibly slow kinetics of phospholipid transfer to or from detergent-solubilized membrane proteins. Independently, continuous recording of the activity of the protein can also be used in some cases to correlate changes in activity with the exchange of a specific phospholipid, as shown here by using the Drs2p/Cdc50p complex, a lipid flippase with specific binding sites for lipids.