Membrane Fusion, Organization, and Dynamics Using Supported Bilayers

描述(由申请人提供)：该项目的长期目标是开发探索生物膜的组织和动态重组的方法。这包括膜内组分之间的相互作用，膜表面之间导致结合、融合和图案形成的相互作用，以及与膜相关的蛋白质的构象变化。脂双层是生物膜共有的基本结构。膜的流动性对生物功能至关重要，它依赖于膜内构象的变化、多组分的侧向结合或聚集，以及改变膜拓扑结构的过程，如EDO-、胞外和融合。这项建议概述了使用已开发的工具来探索膜动力学的这些基本方面的新类型的实验，这些工具已经被开发用于图案化、操纵和成像支持的双层。在下一个赠与期内，重点将放在囊泡融合机制上，利用与支撑双层相连的囊泡，并可在单个囊泡水平上监测其相互作用(目标1)；利用一种新型的成像质谱学，使膜成分分析具有前所未有的横向分辨率、灵敏度和信息含量，从而实现脂质和膜锚定蛋白的横向结合和组织(目标2)；以及设计和制造集成光学/电子设备，从而能够对平面双层进行高精度干涉测量，以探测膜相关蛋白质的构象转变，最初的重点是电压门控离子通道(目标3)。每一个目标都依赖于新的支持脂双层结构和分析方法的发展，这些结构和分析方法可以对生物膜的研究产生广泛的影响。与人类健康相关：所有蛋白质中有很大一部分与膜有关，作为一类，这些蛋白质构成了药物开发的巨大和多样化的靶点。这项建议概述了研究膜和膜相关蛋白的新方法，这些方法可以影响我们对生物功能和组织的理解，以及对生物技术的影响。

DESCRIPTION (provided by applicant): The long-term goals of this project are to develop methods to probe the organization and dynamic reorganization of biological membranes. This includes interactions among the components within membranes, interactions between membrane surfaces that lead to binding, fusion, and pattern formation, and conformational changes of proteins associated with membranes. The lipid bilayer is the basic structure common to biological membranes. Membrane fluidity is critical for biological functions that depend upon conformational changes within membranes, the lateral association or clustering of multiple components, and processes that change membrane topology such as edo- and exocytocis and fusion. This proposal outlines new types of experiments that probe these basic aspects of membrane dynamics using tools that have been developed to pattern, manipulate and image supported bilayers. During the next grant period the focus will be on the mechanism of vesicle fusion, using vesicles that are tethered to supported bilayers and whose interactions can be monitored at the level of individual vesicles (Aim 1); the lateral association and organization of lipids and membrane anchored proteins using a novel type of imaging mass spectrometry that permits membrane composition analysis with unprecedented lateral resolution, sensitivity and information content (Aim 2); and the design and fabrication of an integrated optical/electrical device that will permit high precision interferometry on planar bilayers to probe conformational transitions of membrane-associated proteins, with an initial focus on voltage-gated ion channels (Aim 3). Each aim depends upon the development of new supported lipid bilayer architectures and analytical methods that can have a broad impact on studies of biological membranes. Relevance to human health: A significant fraction of all proteins are associated with membranes, and, as a class, these constitute a huge and diverse target for drug development. This proposal outlines new methods for studying membranes and membrane-associated proteins that can impact our understanding of biological function and organization, as well as impact biotechnology.