Single Molecule Study of Disassembly of SNARE Complex by NSF/α−SNAP

Abstract

In eukaryotic cells, material exchange between distinct organelles within the cytosol is required for many biochemical processes. The phospholipid vesicles containing the materials are transported to the target membrane and merged. All known forms of intracellular membrane fusion or vesicle fusion involve a highly conserved family of proteins termed SNAREs (Soluble N-ethyl maleimide sensitive-factor Attachment Protein Receptors). The assembly and dissociation of SNARE proteins drives membrane fusion. A good example is chemical release in the synapse, which is essential for inter-communication between neurons. Although it has been known that the ATPase activity of NSF (N-ethyl maleimide Sensitive-Factor) is involved in the disassembly of SNARE complex, the molecular details of the reaction are not understood. In order to investigate the molecular mechanism, we applied a fluorescence-based in vitro approach at the single molecule level. SNARE complexes from three neuronal proteins (Syntanxin1a, Snap-25, and Synaptobrevin) were labeled with suitably chosen FRET dye pairs based on the available structural information about the SNARE complex. For the particular labeling pairs, high FRET corresponds to assembled complex, low FRET corresponds to dissociated complex. To test the labels, we used a conventional bulk FRET assay and found a progressive temporal change from high FRET to low FRET states, indicating complex dissociation into each individual protein in a Mg2+, ATP, and α-SNAP dependent manner. Using a single molecule TIRF optical setup, the same reactions were monitored, and likewise show a change in FRET signal corresponding to complex disassembly. We are studying the disassembly reaction for a number of different labeling pairs along the SNARE helical bundle. Single molecule kinetic studies are underway to understand the molecular mechanism of NSF catalyzed SNARE protein disassembly.