Ho C, Stubbs C D
Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
Biophys J. 1992 Oct;63(4):897-902. doi: 10.1016/S0006-3495(92)81671-5.
Evidence has been found for the existence water at the protein-lipid hydrophobic interface of the membrane proteins, gramicidin and apocytochrome C, using two related fluorescence spectroscopic approaches. The first approach exploited the fact that the presence of water in the excited state solvent cage of a fluorophore increases the rate of decay. For 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-palmitoyl-2-[[2-[4-(6-phenyl-trans-1,3,5- hexatrienyl)phenyl]ethyl]carbonyl]-3-sn-PC (DPH-PC), where the fluorophores are located in the hydrophobic core of the lipid bilayer, the introduction of gramicidin reduced the fluorescence lifetime, indicative of an increased presence of water in the bilayer. Since a high protein:lipid ratio was used, the fluorophores were forced to be adjacent to the protein hydrophobic surface, hence the presence of water in this region could be inferred. Cholesterol is known to reduce the water content of lipid bilayers and this effect was maintained at the protein-lipid interface with both gramicidin and apocytochrome C, again suggesting hydration in this region. The second approach was to use the fluorescence enhancement induced by exchanging deuterium oxide (D2O) for H2O. Both the fluorescence intensities of trimethylammonium-DPH, located in the lipid head group region, and of the gramicidin intrinsic tryptophans were greater in a D2O buffer compared with H2O, showing that the fluorophores were exposed to water in the bilayer at the protein-lipid interface. In the presence of cholesterol the fluorescence intensity ratio of D2O to H2O decreased, indicating a removal of water by the cholesterol, in keeping with the lifetime data. Altered hydration at the protein-lipid interface could affect conformation, thereby offering a new route by which membrane protein functioning may be modified.
利用两种相关的荧光光谱方法，已发现证据表明在膜蛋白短杆菌肽和脱辅基细胞色素C的蛋白质 - 脂质疏水界面处存在水。第一种方法利用了这样一个事实，即荧光团激发态溶剂笼中的水会增加衰减速率。对于1,6 - 二苯基 - 1,3,5 - 己三烯（DPH）和1 - 棕榈酰 - 2 - [[2 - [4 - (6 - 苯基 - 反式 - 1,3,5 - 己三烯基)苯基]乙基]羰基] - 3 - sn - 磷脂酰胆碱（DPH - PC），荧光团位于脂质双层的疏水核心中，短杆菌肽的引入降低了荧光寿命，这表明双层中存在更多的水。由于使用了高蛋白：脂质比，荧光团被迫靠近蛋白质疏水表面，因此可以推断该区域存在水。已知胆固醇会降低脂质双层的含水量，并且这种效应在与短杆菌肽和脱辅基细胞色素C的蛋白质 - 脂质界面处也得以维持，这再次表明该区域存在水合作用。第二种方法是利用用重水（D2O）替代水（H2O）所诱导的荧光增强。与H2O缓冲液相比，位于脂质头部基团区域的三甲基铵 - DPH和短杆菌肽内在色氨酸的荧光强度在D2O缓冲液中都更大，这表明荧光团在蛋白质 - 脂质界面的双层中暴露于水。在存在胆固醇的情况下，D2O与H2O的荧光强度比降低，表明胆固醇去除了水，这与寿命数据一致。蛋白质 - 脂质界面处水合作用的改变可能会影响构象，从而为修饰膜蛋白功能提供了一条新途径。
