Structural Analysis of Protein-Membrane Interaction

描述（由申请人提供）：尽管最近取得了巨大进展， 结构生物学，确定膜蛋白的结构 依然困难重重同样地，我们对 蛋白质与膜相互作用的机制。在这里，我建议研究 该过程使用定点自旋标记（SDSL）。SDSL已成为 强大的新技术，用于确定结构和构象动力学， 可溶性和膜蛋白。SDSL不限于特定的蛋白质大小 并可用于在一定条件下真实的实时监测构象变化 生理条件。 这些研究的主要模型系统将是膜联蛋白12，膜联蛋白12是膜联蛋白的一个成员。 膜结合蛋白家族。Annexin 12具有三个明显的 不同的状态：（1）已知结构的水溶性状态，（2） 依赖钙离子的外周膜结合型，正如我们最近发现的， (3)跨膜形式，其是脂质和pH依赖性的，但不需要 Ca2+本提案的目的是研究 外周和整体膜相关状态，并确定因素 调节它们之间可逆的相互转化。另外我们 希望这一结构工作提供一个重要的基础，以评估和 使膜联蛋白的许多膜相关功能合理化。 序列分析表明，其他蛋白质也可以插入细胞膜 使用一种类似膜联蛋白的机制。因为有趣的生理学 的影响，我们将研究这些蛋白质之一的膜相互作用， 雌激素受体α。

DESCRIPTION (provided by applicant): Despite enormous recent progress in structural biology in general, determining the structure of membrane proteins has remained difficult. Similarly, we understand very little about the mechanism by which proteins interact with membranes. Here I propose to study this process using site-directed spin labeling (SDSL). SDSL has become a powerful new technique for determining structure and conformational dynamics in soluble and membrane proteins. SDSL is not limited to a particular protein size and can be used to monitor conformational changes in real time under physiological conditions. The primary model system for these studies will be annexin 12, a member of the annexin family of membrane binding proteins. Annexin 12 has three distinctly different states: (1) a water-soluble state of known structure, (2) a Ca2tdependent, peripherally membrane-bound form, and as we recently discovered, (3) a transmembrane form that is lipid and pH dependent, but does not require Ca2+ It is the goal of this proposal to investigate the structures of the peripheral and integral membrane-associated states and determine the factors that modulate the reversible interconversion between them. In addition, we expect this structural work to provide an important foundation to evaluate and rationalize the numerous membrane-related functions of annexins. Sequence analysis suggests that other proteins also could insert into membranes using an annexin-like mechanism. Because of intriguing physiological implications, we will study the membrane interaction of one of these proteins, the estrogen receptor alpha.