Inter-Leaflet Phospholipid Exchange Impacts the Ligand Density Available for Protein Binding at Supported Lipid Bilayers
Inter-Leaflet Phospholipid Exchange Impacts the Ligand Density Available for Protein Binding at Supported Lipid Bilayers
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DOI：
10.1021/acs.langmuir.2c00526
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发表时间：
2022-05-26
期刊：
影响因子：
3.9
通讯作者：
Harris，Joel M.
中科院分区：
文献类型：
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作者：
Myres，Grant J.;Kitt，Jay P.;Harris，Joel M.
在固-液界面形成的磷脂双分子层作为模拟细胞膜以模拟蛋白质与脂质双分子层束缚的配体的缔合反应而引起了人们的兴趣。尽管了解脂质双层中的配体密度如何影响蛋白质-配体缔合反应的重要性，但将配体修饰的脂质部分与脂质双层中溶液可及配体的绝对密度相关联仍然是界面定量分析中的挑战。在这项工作中，共聚焦拉曼显微镜被用来量化协会的抗生物素IgG与一小部分的生物素化的脂质分散在凝胶相或液晶支持的脂质双层沉积在宽孔二氧化硅表面的内表面上。我们研究的问题，是否小叶间脂质易位有助于人口的解决方案可访问的生物素配体的支持脂质双层的远侧小叶通过比较他们的蛋白质积累反应与分散在脂质单层的配体腈衍生的二氧化硅表面。抗体与分散在凝胶相双层中的生物素配体的结合表现出与IgG在凝胶相单层上的积累相当的生物素覆盖响应，表明凝胶相双层对称性被保留。这一结果与在流体相双层中观察到的抗生物素捕获比在等效生物素级分下制备的流体相单层高60%形成对比。这种增强的蛋白质捕获归因于生物素封端的脂质通过小叶间交换或脂质翻转（液相支持的脂质双层中的一种容易的过程）从双层的表面相关近端小叶转移到溶液暴露的远端小叶。结果表明，谨慎解释的定量研究结果的蛋白质结合分散在流体相磷脂双分子层的脂质束缚配体。
Phospholipid bilayers formed at solid–liquid interfaces have garnered interest as mimics of cell membranes to model association reactions of proteins with lipid bilayer-tethered ligands. Despite the importance of understanding how ligand density in a lipid bilayer impacts the protein–ligand association response, relating the ligand-modified lipid fraction to the absolute density of solution-accessible ligands in a lipid bilayer remains a challenge in interfacial quantitative analysis. In this work, confocal Raman microscopy is employed to quantify the association of anti-biotin IgG with a small fraction of biotinylated lipids dispersed in either gel-phase or liquid-crystalline supported lipid bilayers deposited on the interior surfaces of wide-pore silica surfaces. We examine the question of whether inter-leaflet lipid translocation contributes to the population of solution-accessible biotin ligands on the distal leaflet of a supported lipid bilayer by comparing their protein accumulation response with ligands dispersed in lipid monolayers on nitrile-derivatized silica surfaces. The binding of the antibody to biotin ligands dispersed in gel-phase bilayers exhibited an equivalent biotin coverage response as the accumulation of IgG onto gel-phase monolayers, indicating that gel-phase bilayer symmetry was preserved. This result contrasts with the ∼60% greater anti-biotin capture observed at fluid-phase bilayers compared to fluid-phase monolayers prepared at equivalent biotin fractions. This enhanced protein capture is attributed to biotin-capped lipids being transferred from the surface-associated proximal leaflet of the bilayer to the solution-exposed distal leaflet by the inter-leaflet exchange or lipid flip-flop, a facile process in fluid-phase supported lipid bilayers. The results suggest caution in interpreting the results of quantitative studies of protein binding to lipid-tethered ligands dispersed in fluid-phase phospholipid bilayers.