Protein Recruitment Processes in Asymmetric Bilayer Systems

知识价值。质膜中的脂质和蛋白质通常分布在空间密切相关的不同斑块中，如众所周知的特定膜蛋白与富含鞘脂和胆固醇的脂质斑块（也称为脂筏）的关联。不幸的是，由于表征质膜中功能性脂质异质性的挑战，许多与功能重要的蛋白质-脂质相互作用相关的潜在分子过程仍不清楚。为了克服这些困难，目前的项目正在研究蛋白质-脂质相互作用的特定方面，通过观察木筏模拟液体有序/液体无序相分离，这些相分离跨越双层，只存在于双层的一个小叶中，并确定单层间偶联如何影响选定膜蛋白的隔离和功能。为了实现这一目标，该项目使用定义明确的脂质组成的聚合物系固脂双分子层。本研究包括三个具体目标：(1)探索脂质在单层和双层跨越域的堆积密度；(2)研究糖基磷脂酰肌醇锚定蛋白和跨膜蛋白在单层和双层脂质结构域下的分离；(3)研究这些蛋白质在其天然配体存在下的招募。利用高灵敏度的共聚焦荧光检测系统，通过荧光强度和各向异性分析来确定脂质的堆积密度。膜蛋白的行为将监测与单分子灵敏度使用共聚焦荧光强度分析和宽视场单分子荧光显微镜。通过在单个蛋白质水平上分析分子过程，该方法非常适合探索质膜中蛋白质-脂质相互作用的几个鲜为人知的过程。例如，脂质的跨双层偶联对脂筏中膜蛋白的隔离的影响将被阐明。此外，关于不对称双层组成和筏结构域的叶间偶联在蛋白质相互作用和蛋白质募集过程中的作用的新观点应该作为这些研究的结果被期待。更广泛的影响。该项目提供了一个强大的实验和训练工具来研究各种膜蛋白在良好定义的环境中的分子过程。该研究项目的跨学科特点将为研究生和本科生提供优秀的培训，从而为他们在传统科学、材料科学、工程和医学之间的新兴领域的职业生涯做好准备。由于位于城市校园，有大量的少数民族学生，首席研究员将继续致力于少数民族学生的教育，并将扩大在高中水平的推广活动（实习，高中科学/数学夏令营），并通过机构纳米成像中心。研究成果也将整合到几个特定的本科和研究生水平的课程（物理化学，仿生化学和生物材料科学）。

Intellectual merit. Lipids and proteins in the plasma membrane are often distributed in widely differing patches of close spatial correlation, as exemplified by the well known association of particular membrane proteins to lipid patches enriched in sphingolipids and cholesterol, also known as lipid rafts. Unfortunately, many of the underlying molecular processes related to functionally important protein-lipid interactions remain unclear due to the challenges involved in characterizing functional lipid heterogeneities in plasma membranes. To overcome these difficulties, the current project is investigating specific aspects of protein-lipid interactions by looking at raft-mimicking liquid-ordered/liquid-disordered phase separations which span bilayers and which only exist in one leaflet of a bilayer, and determining how inter-monolayer coupling affects the sequestration and functionality of select membrane proteins. To achieve this objective, the project uses polymer-tethered lipid bilayers of well-defined lipid compositions. The research includes three specific aims, which are: (1) to explore the packing density of lipids in both monolayer and bilayer-spanning domains; (2) to study the sequestering of select glycosylphosphatidylinositol-anchored proteins and membrane-spanning proteins in the presence of both monolayer and bilayer-spanning lipid domains; and (3) to investigate the recruitment of these proteins in the presence of their native ligands. The packing density of lipids will be determined by fluorescence intensity and anisotropy analysis using a highly sensitive confocal fluorescence detection system. The behavior of membrane proteins will be monitored with single molecule sensitivity using confocal fluorescence intensity analysis and wide-field single molecule fluorescence microscopy. By enabling the analysis of molecular processes at the single protein level, the approach is well suited to explore several of the poorly understood processes of protein-lipid interplay in plasma membranes. For example, the impact of transbilayer coupling of lipids on the sequestering of membrane proteins in lipid rafts will be elucidated. Furthermore, new perspectives about the roles of asymmetric bilayer composition and inter-leaflet coupling of raft domains on protein-protein interactions and protein recruitment processes should be anticipated as an outcome of these investigations.Broader impacts. The project provides a powerful experimental and training tool to study molecular processes of a wide variety of membrane proteins in well-defined environments. The interdisciplinary character of the research project will provide excellent training for graduate and undergraduate students, thus preparing them for professional careers in emerging areas at the interface between traditional sciences, materials science, engineering, and medicine. Being located at an urban campus with a significant minority student population, the principal investigator will remain committed to minority student education and will expand outreach activities at the high-school level (internships, high-school science/math summer camp) and through the institutional Nanoscale Imaging Center. Research results will also be integrated into several specific undergraduate- and graduate-level courses (physical chemistry, biomimetic chemistry, and biomaterials science).