FTIR and fluorescence studies of interactions of synaptic fusion proteins in polymer-supported bilayers

Single planar phospholipid bilayers supported on soft polymer (poly(ethylene glycol), PEG) cushions have served as useful models for biological membranes. Integral membrane proteins can be successfully reconstituted in covalently tethered PEG3400-supported bilayers (=DPS-supported bilayers) where they bind their natural ligands as in biological membranes and where they exhibit almost normal physiological lateral diffusion properties. Here, we have characterized DPS-supported bilayers by polarized attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. The bilayers are not significantly perturbed by the polymer, and the vibrational bands of PEG do not interfere with the interpretation of several major lipid and protein bands, clearing the way to study protein secondary structure and lipidprotein interactions in DPS-supported lipid bilayers by ATR-FTIR spectroscopy. We have also reconstituted v- and t-SNARE proteins and formed membrane-bound SNARE complexes in this model system. Membrane-bound v-SNAREs (VAMP2 or synaptobrevin2) are unstructured, and membrane-bound t-SNAREs (syntaxin 1A/SNAP-25) are predominantly a-helical. v-SNAREs, but not t-SNAREs, exchange readily between the polymer-supported bilayer and lipid vesicles in solution, suggesting that v-SNAREs do not possess a classical stable transmembrane anchor at their C-terminus. SNARE complex formation between the polymer-supported membrane and lipid vesicles does not induce detectable additional alpha-helical secondary structure in this system.