Biophysical Society Thematic Meeting

Liposomes, Exosomes, and Virosomes: From Modeling Complex

Membrane Processes to Medical Diagnostics and Drug Delivery

Poster Abstracts

35-POS

Board 18

Probing Conformational States of the SNARE Fusion Machinery Using Supported Lipid

Bilayers and Site Directed FLIC Microscopy

Volker Kiessling

, Binyong Liang, Lukas K. Tamm.

University of Virginia, Charlottesville, VA, USA.

The fusion of synaptic vesicles with the presynaptic membrane is a fast and highly regulated

process that is catalyzed by the neuronal SNAREs synaptobrevin-2 (VAMP-2), SNAP-25, and

syntaxin-1a (Syx1a). In addition to the SNAREs a number of protein players that regulate the

fusion process have been identified and characterized. Despite tremendous progress in this field

we are still missing a molecular timeline that leads from docking of synaptic vesicles to the

plasma membrane, to priming of the fusion machinery and eventually to fusion of the two

membranes once an action potential reaches the synaptic terminal. One of the difficulties in

approaching these questions arises from the fact that the lipid environment plays an active

previously under-appreciated role in this protein driven process.

Reconstituting SNAREs into supported lipid bilayers allows us to characterize the state of the

SNARE and regulatory proteins in different defined lipid environments. Using site-directed

fluorescence interference contrast (sd-FLIC) microscopy we measure distances of specific

residues in Syx1a and VAMP-2 from the membrane surface – and thus conformational

transitions of these proteins – at nm-resolution during proposed steps of the priming pathway.

Here we report how the conformational states of Syx1a and VAMP-2 change upon binding to

regulatory proteins such as Munc18.