Institut de Nanociència i Nanotecnologia IN²UB, Parc Científic de Barcelona, 08028 Barcelona, Spain.
J Phys Chem B. 2012 Mar 1;116(8):2438-45. doi: 10.1021/jp2105665. Epub 2012 Feb 17.
Förster resonance energy transfer (FRET) is a powerful method for the characterization of membrane proteins lipid selectivity. FRET can be used to quantify distances between a single donor and a single acceptor molecule; however, for FRET donors and acceptors scattered in the bilayer plane, multiple donor-acceptor pairs and distances are present. In addition, when studying protein/lipid selectivity, for a single tryptophan used as a donor; several lipid acceptors may be located at the boundary region (annular lipids) of the protein. Therefore, in these experiments, a theoretical analysis based on binomial distribution of multiple acceptors around the membrane proteins is required. In this work, we performed FRET measurements between single tryptophan lactose permease (W151/C154G LacY) of Escherichia coli and pyrene-labeled phospholipids (Pyr-PE, Pyr-PG, and Pyr-PC) reconstituted in palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-choline, and 1,2-dioleoyl-sn-glycero-3-phospho-choline at 25 and 37 °C. To increase the sensitivity of the method and to ascertain the lipid selectivity for LacY, we reconstituted the protein in the pure phospholipids doped with 1.5% of labeled phospholipids. From fitting the theoretical model to the experimental FRET efficiencies, two parameters were calculated: the probability of a site in the annular ring being occupied by a labeled pyrene phospholipid and the relative association constant between the labeled and unlabeled phospholipids. The experimental FRET efficiencies have been interpreted taking into account the particular folding of the protein in each phospholipid matrix. Additional information on the annular lipid composition for each system has been obtained by exciting W151/C154G LacY and monitoring the emission intensities for monomer and excimer of the pyrene spectra. The results obtained indicate a higher selectivity of LacY for PE over PG and PC and pointed to a definite role of the acyl chains in the overall phospholipid-protein interaction.
Förster 共振能量转移（FRET）是一种用于表征膜蛋白脂质选择性的强大方法。FRET 可用于量化单个供体和单个受体分子之间的距离；然而，对于分散在双层平面中的 FRET 供体和受体，存在多个供体-受体对和距离。此外，在研究蛋白质/脂质选择性时，对于用作供体的单个色氨酸，几个脂质受体可能位于蛋白质的边界区域（环形脂质）。因此，在这些实验中，需要基于膜蛋白周围的多个受体的二项式分布进行理论分析。在这项工作中，我们在大肠杆菌单色氨酸乳糖透酶（W151/C154G LacY）和芘标记的磷脂（Pyr-PE、Pyr-PG 和 Pyr-PC）之间进行了 FRET 测量，这些磷脂在棕榈酰-2-油酰-sn-甘油-3-磷酸乙醇胺、1-棕榈酰-2-油酰-sn-甘油-3-[磷酸-rac-(1-甘油基)]（钠盐）、1-棕榈酰-2-油酰-sn-甘油-3-磷酸胆碱和 1,2-二油酰基-sn-甘油-3-磷酸胆碱中重建，温度为 25 和 37°C。为了提高方法的灵敏度并确定 LacY 的脂质选择性，我们在含有 1.5%标记磷脂的纯磷脂中重建了蛋白质。通过将理论模型拟合到实验 FRET 效率，计算了两个参数：环形环中一个位点被标记芘磷脂占据的概率和标记与未标记磷脂之间的相对结合常数。考虑到每种磷脂基质中蛋白质的特定折叠，对实验 FRET 效率进行了解释。通过激发 W151/C154G LacY 并监测芘光谱的单体和激态发射强度，获得了每个系统中环形脂质组成的附加信息。结果表明，LacY 对 PE 的选择性高于 PG 和 PC，并且酰基链在整个磷脂-蛋白质相互作用中起着明确的作用。
