Protein Recruitment Processes in Asymmetric Bilayer Systems

不对称双层系统中的蛋白质招募过程

• 批准号: 0920134
• 负责人: Christoph Naumann
• 金额: $ 44.25万
• 依托单位: Indiana University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-15 至 2013-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0920134&HistoricalAwards=false
• 关键词: Protein; Recruitment; Processes; Asymmetric; Bilayer

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).

智力上的优点。质膜中的脂质和蛋白质通常分布在具有密切空间相关性的广泛不同的斑块中，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）的结合所例示的。不幸的是，许多潜在的分子过程相关的功能重要的蛋白质-脂质相互作用仍然不清楚，由于在质膜中的功能脂质异质性的特征所涉及的挑战。为了克服这些困难，目前的项目正在研究蛋白质-脂质相互作用的具体方面，通过观察跨越双层并且仅存在于双层的一个小叶中的筏模拟液体有序/液体无序相分离，并确定单层间耦合如何影响选择膜蛋白的螯合和功能。为了实现这一目标，该项目使用聚合物拴系的脂质双层明确的脂质组成。该研究包括三个具体目标，即：（1）探索单层和跨双层结构域中脂质的堆积密度;（2）研究选择的糖基磷脂酰肌醇锚定蛋白和跨膜蛋白在单层和跨双层脂质结构域存在下的螯合作用;以及（3）研究这些蛋白在其天然配体存在下的募集。脂质的堆积密度将通过使用高灵敏度共聚焦荧光检测系统的荧光强度和各向异性分析来确定。将使用共聚焦荧光强度分析和宽视野单分子荧光显微镜，以单分子灵敏度监测膜蛋白的行为。通过在单个蛋白质水平上分析分子过程，该方法非常适合于探索质膜中蛋白质-脂质相互作用的几个知之甚少的过程。例如，将阐明脂质的跨双层偶联对脂质筏中膜蛋白的螯合的影响。此外，新的观点的作用，不对称的双层组成和叶间耦合筏结构域上的蛋白质-蛋白质相互作用和蛋白质招募过程中，应预期作为这些调查的结果。该项目提供了一个强大的实验和培训工具，以研究各种膜蛋白在明确定义的环境中的分子过程。该研究项目的跨学科性质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。位于一个城市校园与一个显着的少数民族学生人口，主要研究者将继续致力于少数民族学生的教育，并将扩大外展活动在高中水平（实习，高中科学/数学夏令营），并通过机构纳米成像中心。研究成果也将被整合到几个特定的本科和研究生课程（物理化学，仿生化学和生物材料科学）。