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.