Dumas F, Tocanne J F, Leblanc G, Lebrun M C
Institut de Pharmacologie et Biologie Structurale du CNRS,205, Route de Narbonne F-31077 Toulouse Cedex, France.
Biochemistry. 2000 Apr 25;39(16):4846-54. doi: 10.1021/bi992634s.
The structural and functional consequences of a mismatch between the hydrophobic thickness d(P) of a transmembrane protein and that d(L) of the supporting lipid bilayer were investigated using melibiose permease (MelB) from Escherichia coli reconstituted in a set of bis saturated and monounsaturated phosphatidylcholine species differing in acyl-chain length. Influence of MelB on the midpoint gel-to-liquid-phase transition temperature, T(m), of the saturated lipids was investigated through fluorescence polarization experiments, with 1,6-diphenyl-1,3,5-hexatriene as the probe, for varying protein/lipid molar ratio. Diagrams in temperature versus MelB concentration showed positive or negative shifts in T(m) with the short-chain lipids DiC12:0-PC and DiC14:0-PC or the long-chain lipids DiC16:0-PC and DiC18:0-PC, respectively. Theoretical analysis of the data yielded a d(L) value of 3.0 +/- 0.1 nm for the protein, similar to the 3.02 nm estimated from hydropathy profiles. Influence of the acyl chain length on the carrier activity of MelB was investigated in the liquid phase, using the monounsaturated PCs. Binding of the sugar to the transporter showed no dependence on the acyl chain length. In contrast, counterflow and Deltapsi-driven experiments revealed strong dependence of melibiose transport on the lipid acyl chain length. Similar bell-shaped transport versus acyl chain length profiles were obtained, optimal activity being supported by diC16:1-PC. On account of a d(P) value of 2.65 nm for the lipid and of various local constraints which would all tend to elongate the acyl chains in contact with the protein, one can conclude that maximal activity was obtained when the hydrophobic thickness of the bilayer matched that of the protein.
利用重组于一组酰基链长度不同的双饱和和单不饱和磷脂酰胆碱中的大肠杆菌蜜二糖通透酶（MelB），研究了跨膜蛋白的疏水厚度d(P)与支持性脂质双层的疏水厚度d(L)不匹配所产生的结构和功能后果。通过荧光偏振实验，以1,6 - 二苯基 - 1,3,5 - 己三烯为探针，针对不同的蛋白质/脂质摩尔比，研究了MelB对饱和脂质的中点凝胶 - 液相转变温度T(m)的影响。温度与MelB浓度的关系图显示，对于短链脂质二肉豆蔻酰磷脂酰胆碱（DiC12:0 - PC）和二棕榈酰磷脂酰胆碱（DiC14:0 - PC）或长链脂质二硬脂酰磷脂酰胆碱（DiC16:0 - PC）和二油酰磷脂酰胆碱（DiC18:0 - PC），T(m)分别出现正向或负向偏移。对数据的理论分析得出该蛋白的d(L)值为3.0±0.1 nm，与根据亲水性图谱估计的3.02 nm相似。在液相中使用单不饱和磷脂酰胆碱研究了酰基链长度对MelB载体活性的影响。糖与转运蛋白的结合显示出对酰基链长度没有依赖性。相反，反向流动和Δψ驱动的实验表明蜜二糖转运强烈依赖于脂质酰基链长度。获得了类似的钟形转运与酰基链长度关系曲线，二油酰磷脂酰胆碱（diC16:1 - PC）支持最佳活性。鉴于脂质的d(P)值为2.65 nm以及各种局部限制因素都会倾向于使与蛋白质接触的酰基链伸长，因此可以得出结论，当双层的疏水厚度与蛋白质的疏水厚度匹配时可获得最大活性。
