Annexins form a group of homologous cytosolic proteins that bind phospholipid membranes in a calcium-dependent manner. They have been implicated in a variety of cell functions, including secretion, fusion, and signal transduction. Annexins, which typically have molecular weights of 35kDa or 68kDa, exhibit a common canonical sequence of about 70 residues, repeated four or eight times. These repeats do not fit the pattern predicted for "E-F hand" domains characteristic of the calmodulin superfamily of calcium-binding proteins, which do not bind phospholipids. As such, annexins represent a novel group of calcium-mediating proteins. The long-term goal of this research is to develop a molecular mechanistic model that may be generally applicable to annexins and other calcium- dependent phospholipid-binding proteins. the specific aims addressed in this proposal are the structural characterization of annexin V, a representative annexin family member, and its interactions with calcium and phospholipid ligands through X-ray crystallography and Raman spectroscopy. The crystal structure(s) will provide a detailed molecular model of the protein, while the spectroscopic studies will probe the effects of the protein-phospholipid binding in solution. Raman spectroscopy will provide a bridge between crystal and solution experiments, where samples can be in either form. Large single crystals of Ca2+-bound annexin V have been grown that are suitable for X-ray crystallographic structure determination. They diffract to at least 2.2A resolution and belong to the R3 space group, with hexagonal unit cell dimensions of a=b=156.8A and c=36.9A. To solve the phase problem, a multiple isomorphous replacement (MIR) or iterative single isomorphous replacement (ISIR) approach will be used. Attempts will be used. Attempts will be made to grow and analyze annexin V crystals in other liganded states, such as the Ca2+-free form or with a soluble phospholipid ligand bound. Raman spectroscopy provides detailed information on protein and lipid conformation, and will be used to study ligand-induced conformational changes in annexin V. A high-quality preliminary Raman spectrum of annexin V has been obtained. This spectroscopic technique will be used to evaluate the effects of annexin-phospholipid interactions on the protein structure, and the bound phospholipid, if a suitable reconstitution system can be developed. Peak assignments will be facilitated by the use of phospholipids with fully deuterated hydrocarbon tails.