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Synaptotagmin 1 (syt1) is a synaptic vesicle protein believed to act as the Ca2+ sensor for neuronal exocytosis. It consists of one N-terminal transmembrane helical segment and two C2 domains (C2A and C2B) that are connected by a short, flexible linker. The calcium binding loops of each C2 domain coordinate Ca2+ ions and bind anionic phospholipids. Syt1 also interacts with the neuronal SNARE proteins, which may play a role in the fusion process. We are characterizing the structure of syt1 both in its aqueous and membrane bound states and bound to the soluble core SNARE complex. Double cysteine mutations were engineered into a water soluble fragment of syt1 C2A-C2B and derivatized with the methanethiosulfonate spin label. Four-pulse DEER was used to obtain distance measurements between C2A and C2B in solution, with membranes, and bound to the soluble SNARE complex. The DEER-derived distances were used as restraints in a simulated annealing routine. The predominant structure is one where the C2 domains are separated by about 40 Angstroms and are oriented anti-parallel so that their Ca2+-binding loops are positioned in opposite directions. Broad distance distributions are obtained by DEER, and indicate structural heterogeneity which may be the result of the flexible linker segment joining the two domains. This structural arrangement does not change when the protein is bound to membranes or the soluble SNARE complex. Furthermore, C2A-C2B is shown to bridge bilayers, which is mediated by multiple contacts of the positive charged regions of the C2B domain and the anti-parallel orientation of C2A and C2B. The result suggests that one role for syt1 in fusion is to bridge across the vesicle and plasma membrane surfaces in a Ca2+-dependent manner. The work was supported by NIGMS grant GM 72694.