ANNEXIN CRYSTAL STRUCTURES AND MEMBRANE INTERACTIONS

作用于膜-水界面的蛋白质在独特的环境中起作用，其分子机制代表了细胞生物学的当前前沿。 膜联蛋白是一个结构上同源的界面蛋白大家族，以钙依赖性方式与膜结合。 在各种来源中发现大量（总细胞蛋白的1- 2%），膜联蛋白广泛分布于人类和其他真核生物中。 近年来，膜联蛋白V已经成为膜联蛋白家族和其他外周膜蛋白，特别是那些结合钙的膜蛋白的机制范例。 对于结构生物学家来说，膜联蛋白V提供了一个不寻常的研究机会，因为该蛋白质可以采用稳定的水溶性或膜结合形式，易于表征。 该研究项目的长期目标是开发膜联蛋白在膜上行为的综合图像，并通过表征膜联蛋白与膜组分的相互作用和对膜特性的影响来探索膜联蛋白的生物学作用。 在拟议的研究中，我们将继续关注膜联蛋白V与膜的相互作用。 我们还将研究一个新的领域，膜联蛋白表面面向远离膜的水环境。 该表面含有N-末端，其被认为赋予个体膜联蛋白功能。 在这种情况下，我们将扩大我们的视野，进一步包括膜联蛋白IV，膜联蛋白V的一个密切的结构同源物，但它是不同的几个关键属性，包括被蛋白激酶C磷酸化的能力，促进囊泡聚集，抑制氯离子通道活性。 这些特性与膜联蛋白IV分子的N-末端区域最密切相关。 我们还将介绍对贾第虫素的研究，贾第虫素是一种来自原生动物蓝氏贾第虫的“原始”膜联蛋白，它似乎有助于将寄生虫附着在其宿主上。 在所研究的系统中的一个共同主题是膜联蛋白-膜附着是多个水平上的细胞组织的主要组成部分，影响蛋白质-膜、膜-膜、蛋白质-蛋白和细胞-细胞相互作用。对于拟议的研究，我们将使用一套互补的方法，包括X射线晶体学，定点诱变，光谱学和其他生物物理或生物化学方法。 我们预计，这些综合研究将导致膜联蛋白的共同特征，主要是钙膜结合，以及赋予个别膜联蛋白不同功能的功能的理解。 总之，这些研究的结果将增加我们对界面蛋白的理解，为膜联蛋白在膜上的作用提供统一的结构基础，并提出膜联蛋白功能的结构基础，因为它们与人类健康和疾病有关。

Proteins that act at the membrane-aqueous interface operate in a unique environment, and their molecular mechanisms represent a current frontier of cell biology. Annexins comprise a large family of structurally homologous, interfacial proteins that bind membranes in a calcium-dependent manner. Found in large amounts (1-2 percent total cell protein) in various sources, annexins are widely distributed in humans and other eukaryotes. In recent years, annexin V has emerged as a mechanistic paradigm for the annexin family and other peripheral membrane proteins, particularly those that bind calcium. For structural biologists, annexin V presents an unusual opportunity for study since the protein can adopt stable water-soluble or membrane-bound forms that are amenable to characterization. The long-term goal of this research project is to develop an integrated picture of annexin behavior at the membrane, and to probe the biological roles of annexins by characterizing their interactions with membrane components and influence on membrane properties. In the proposed studies, we will continue to focus on annexin V interactions with the membrane. We will also investigate a new area, the annexin surface facing away from the membrane toward the aqueous milieu. This surface contains the N-terminus, which is believed to confer individual annexin function. In this context, we will broaden our view further to include annexin IV, a close structural homolog of annexin V but which is distinct in several key properties, including the ability to be phosphorylated by protein kinase C, promote vesicle aggregation, and inhibit chloride channel activity. These properties are most closely associated with the N-terminal region of the annexin IV molecule. We will also introduce studies of a-giardin, a "primitive" annexin from the protozoan Giardia lamblia, which appears to assist in attaching the parasite to its host. A common theme in the systems under investigation is that annexin-membrane attachment is a major component of cellular organization on multiple levels, influencing protein-membrane, membrane-membrane, protein-protein, and cell-cell interactions. For the proposed studies, we will use a complementary set of approaches including x-ray crystallography, site-directed mutagenesis, spectroscopy and other biophysical or biochemical methods. We anticipate that these combined studies will lead to an understanding of both the common features of annexins, primarily calcium-membrane binding, and the features that endow individual annexins with distinct functions. Taken together, the results from these studies will add to our understanding of interfacial proteins, provide a unifying structural basis for annexin action at the membrane, and propose structural bases for annexin functions as they relate to human health and disease.