Newly synthesized proteins are delivered to final destinations by way of vesicular transport. Assembly of the complex between target membrane (t-) SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) and vesicle-associated (v-) SNARE is essential for docking and membrane fusion. The goal of this project is to elucidate the process of SNARE assembly and the mechanism of membrane fusion involved in yeast protein trafficking. SNAREs are amphipathic integral membrane proteins that offer quite a challenge to x-ray crystallography and NMR. The present project utilizes site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) that has proven powerful in determining structures and topologies of integral membrane proteins. In this method, native residues are substituted to cysteines and modified with a nitroxide spin label. Recent advances in EPR spectroscopy allow the measurement of membrane immersion depths as well as distances between pairs of positions in the range of 6-50 Angstroms. Data analysis and computer modeling on a sufficiently large set of distances give a three-dimensional model of the protein structure at backbone resolution. SNARE assembly proceeds in multiple steps. In this project, structures and membrane topologies of individual full-length SNARE proteins, their assembly intermediates, and the final complex are investigated with SDSL EPR. It is widely believed that SNAREs are the minimal fusion machinery. Thus, EPR data will provide insights into the mechanism of SNARE-induced membrane fusion. Remarkably, all SNAREs involved in vesicular transports are highly conserved. Thus, what is learned from yeast SNAREs will have implications for other systems.
