Merino Sandra, Domènech Oscar, Viñas M, Montero M Teresa, Hernández-Borrell Jordi
Departament de Fisicoquímica and Química Física, Universitat de Barcelona, E-08028 Barcelona, Spain.
Langmuir. 2005 May 10;21(10):4642-7. doi: 10.1021/la047102d.
The membrane transport protein lactose permease (LacY), a member of the major facilitator superfamily containing 12 membrane-spanning segments connected by hydrophilic loops, was reconstituted in liposomes whose composition was 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol in a 3:1 molar ratio. The structural order of the lipid membranes, in the presence and absence of LacY, was assessed using steady-state fluorescence anisotropy. The features of the anisotropy curves obtained with 1,6-phenyl-1,3,5-hexatriene and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate suggest a surface effect of LacY on the membranes. Atomic force microscopy imaging of supported planar bilayers (SPBs) deposited onto mica was used to examine the effect of LacY on the nanostructure of the phospholipid matrix. Two separated domains were observed in SPBs formed from pure phospholipid mixture. Protein assemblies segregated from the rest of the matrix were observed after the extension of proteoliposomes. The effect of the protein on the electrostatic surface potential of the bilayer was also examined using a fluorescent pH indicator, 4-heptadecyl-7-hydroxycoumarin. Changes in surface potential were enhanced in the presence of the substrate (i.e., lactose). Taken together the results indicate that LacY is segregated into the phospholipid matrix and has moderate effects on the acyl chain order of the bilayers. The changes in surface electrical properties of the bilayers suggest a role for the phospholipid headgroups in proton transfer to the amino acids involved in substrate translocation.
膜转运蛋白乳糖通透酶（LacY）是主要易化子超家族的成员，包含12个跨膜片段，由亲水性环连接，它被重组到脂质体中，脂质体的组成是1-棕榈酰-2-油酰-sn-甘油-3-磷酸乙醇胺和1-棕榈酰-2-油酰-sn-甘油-3-磷酸甘油，摩尔比为3:1。在有和没有LacY的情况下，使用稳态荧光各向异性评估脂质膜的结构顺序。用1,6-苯基-1,3,5-己三烯和1-(4-三甲基铵苯基)-6-苯基-1,3,5-己三烯对甲苯磺酸盐获得的各向异性曲线特征表明LacY对膜有表面效应。使用原子力显微镜对沉积在云母上的支持平面双层（SPB）进行成像，以检查LacY对磷脂基质纳米结构的影响。在由纯磷脂混合物形成的SPB中观察到两个分离的区域。在蛋白脂质体延伸后，观察到与基质其余部分分离的蛋白质聚集体。还使用荧光pH指示剂4-十七烷基-7-羟基香豆素检查了该蛋白对双层静电表面电位的影响。在存在底物（即乳糖）的情况下，表面电位的变化增强。综合这些结果表明，LacY被隔离到磷脂基质中，对双层的酰基链顺序有中等程度的影响。双层表面电学性质的变化表明磷脂头部基团在质子转移到参与底物转运的氨基酸中起作用。
