ANNEXIN CRYSTAL STRUCTURES AND MEMBRANE INTERACTIONS

作用于膜-水界面的蛋白质在独特的环境中运作，其分子机制代表了当前细胞生物学的前沿。膜联蛋白包括一个结构上同源的界面蛋白大家族，以钙依赖的方式结合膜。膜联蛋白在各种来源中大量存在（占细胞总蛋白的1- 2%），广泛分布于人类和其他真核生物中。近年来，膜联蛋白V已成为膜联蛋白家族和其他外周膜蛋白，特别是结合钙的外周膜蛋白的机制范例。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，因为该蛋白可以采用稳定的水溶性或膜结合形式，易于表征。本研究项目的长期目标是发展膜联蛋白在膜上行为的整体图景，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区域，即膜联蛋白表面远离膜朝向水环境。这个表面包含n端，它被认为赋予单独的膜联蛋白功能。在这种情况下，我们将进一步扩大我们的视野，包括膜联蛋白IV，它是膜联蛋白V的结构同源物，但在几个关键特性上是不同的，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。这些性质与膜联蛋白IV分子的n端区域密切相关。我们还将介绍对贾第鞭毛虫的研究，这是一种来自原生动物贾第鞭毛虫的“原始”膜联蛋白，它似乎有助于寄生虫附着在宿主上。所研究的系统中的一个共同主题是膜联-膜附着是细胞组织在多个层面上的主要组成部分，影响蛋白质-膜、膜-膜、蛋白质-蛋白质和细胞-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化方法。我们预计这些联合研究将导致对膜联蛋白共同特征的理解，主要是钙膜结合，以及赋予单个膜联蛋白不同功能的特征。综上所述，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，并提出与人类健康和疾病相关的膜联蛋白功能的结构基础。

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.