The mechanisms by which cells distribute materials among intracellular organelles are of fundamental importance to cell biology, yet very little is known about the structures and conformational dynamics of the proteins that mediate intracellular trafficking and secretion. Proposed studies address this gap through x-ray crystallographic and biophysical studies of SNARE proteins, which are centrally involved in the docking and/or fusion of intracellular transport vesicles. These studies focus on two particular sets of SNARE proteins: one set is required in yeast for the fusion of Golgi-derived vesicles with the plasma membrane; the second set, homologous to the first, is required in neurons for the Ca2+-triggered fusion of synaptic vesicles with the presynaptic plasma membrane. The x-ray crystal structure of a domain of the neuronal SNARE protein syntaxin will be determined. This structure should provide a blueprint for designing experiments to probe the apparent regulatory role of this domain in SNARE complex assembly. Crystallization studies are proposed on a modified complex of the neuronal SNAREs syntaxin, SNAP-25, and VAMP/synaptobrevin. This complex, produced in large quantities by co-expression in insect cells, is highly soluble and monodisperse and should represent an improved substrate for crystallization. The homologous yeast complex has also been engineered to have properties favorable for crystallization. Diffraction-quality crystals of one or both complexes will be used for x-ray structural studies. The assembly and disassembly of SNARE complexes, processes accompanied by striking conformational changes, are likely to be important for the functioning of the SNARE proteins and regulated by other proteins. Biophysical techniques will be used to characterize yeast SNARE complex formation in structural, energetic, and kinetic terms. The resulting quantitative framework will be used to evaluate the roles of known and suspected regulatory proteins in vitro. These studies may lend insight into protein-mediated membrane fusion, which is of critical importance not only in intracellular trafficking but also in many other biological processes including fertilization and infection by enveloped viruses such as HIV-1 and influenza.
| Munson, Mary; Hughson, Frederick M (2002) Conformational regulation of SNARE assembly and disassembly in vivo. J Biol Chem 277:9375-81 |
| Lerman, J C; Robblee, J; Fairman, R et al. (2000) Structural analysis of the neuronal SNARE protein syntaxin-1A. Biochemistry 39:8470-9 |
