Structural Analysis of Protein-Membrane Interaction

描述(由申请人提供)：尽管最近在 一般结构生物学，确定膜蛋白的结构 一直都很困难。同样地，我们对此也知之甚少 蛋白质与膜相互作用的机制。在这里，我提议研究 这一过程使用了定点定向自旋标记(SDSL)。SDSL已成为 用于确定结构和构象动力学的强有力的新技术 可溶性蛋白和膜蛋白。SDSL不限于特定的蛋白质大小 并可用来实时监测在 生理条件。 这些研究的主要模型系统将是Annexin 12，它是 膜联蛋白家族的膜结合蛋白。Annexin 12有三个明显的特征 不同的状态：(1)已知结构的水溶性状态，(2)a 依赖于钙离子，外周膜结合的形式，正如我们最近发现的那样， (3)依赖于脂质和pH的跨膜形式，但不需要 这项建议的目的是研究钙离子的结构 外周和整体膜相关状态和决定因素 这调节了它们之间的可逆相互转化。此外，我们 期待这项结构性工作为评估和评估提供重要基础 使膜联蛋白的众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到膜中 使用一种类似膜联蛋白的机制。因为耐人寻味的生理学 暗示，我们将研究其中一种蛋白质的膜相互作用， 雌激素受体阿尔法。

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.