MICROSTRUCTURAL HETEROGENEITY IN MEMBRANES

这项提议的目的是测试和应用一种新技术(荧光 寿命异质性分析)，用于识别 膜结构的微观非均质性。生命的力量 异质性分析是其潜在的检测灵敏度 不以热力学形式出现的短寿命膜微区 截然不同的阶段。这项技术的适用性首先将是 在简单模型膜系统中进行评估，在该系统中 异质性得到了很好的确立。然后，作为最初的应用程序，这 方法将被用来检验源自 上一次授权期：1)跨膜蛋白同时订购和 破坏局部脂质环境；2)胆固醇诱导 流体脂双分子层中量热无法检测到的局部区域。 新的应用将使用寿命分析来检测微结构 被认为与两个关键的细胞过程有关，膜融合 磷脂跨膜迁移。除了荧光之外 寿命分析、适当的热力学技术(扫描 量热法、稳态探针荧光、化学标记、 冷冻断口电子显微镜)将用于检查双分子层 在定义明确的模型中与这些事件相关联的结构特征 系统。在聚乙二醇膜融合的情况下，这些 测量将检验不同结构的假设 特征与以前密不可分的两个关键因素相联系 融合过程中的中间态：双层闭合对置和 双层失稳。在阳离子诱导的情况下 磷脂酰甘油跨双层重分布，结果将测试两个 假设：1)跨双分子层的再分配涉及两个组分 金属离子与脂质双层的相互作用及2)这种相互作用 在脂双层中诱导出倒置胶束样局部结构。

The aim of this proposal is to test and apply a new technique (fluorescence lifetime heterogeneity analysis) for identifying the existence of microheterogeneity in membrane structure. The power of lifetime heterogeneity analysis is its potential sensitivity for detecting short-lived membrane microdomains which do not appear as thermodynamically distinct phases. The applicability of this technique will first be evaluated in simple model membrane systems in which structural heterogeneity is well established. Then, as initial applications, this method will be used to test two hypotheses stemming from work of the previous grant period: 1) that a trans-membrane protein both orders and disorders its local lipid environment, and 2) that cholesterol induces calorimetrically undetectable local domains in fluid lipid bilayers. New applications will use lifetime analysis to detect microstructures thought to be associated with two key cellular processes, membrane fusion and phospholipid trans-membrane migration. In addition to fluorescence lifetime analysis, appropriate thermodynamic techniques (scanning calorimetry, steady-state probe fluorescence, chemical labeling, freeze-fracture electron microscopy) will be used to examine bilayer structural features associated with these events in well-defined, model systems. In the case of polyethyleneglycol-induced membrane fusion, these measurements will test the hypothesis that different structural characteristics are associated with two crucial and previously inseparable intermediate states in the fusion process: bilayer close apposition and bilayer destabilization. In the case of cation-induced phosphatidylglycerol transbilayer redistribution, results will test two hypotheses: 1) transbilayer redistribution involves a two-component interaction between a metal ion and a lipid bilayer and 2) this interaction induces inverted micelle-like local structures in the lipid bilayer.