The ability of neutral polymer cushions to support neutral lipid bilayers for the incorporation of mobile transmembrane proteins was investigated. Polyacrylamide brush layers were grown on fused silica using atom-transfer radical polymerization to provide polymer layers of 2.5-, 5- and 10-nm thickness. Lipid bilayers composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine) were formed by vesicle fusion onto bare fused silica and onto each of the polyacrylamide layers. Bilayer fluidity was assessed by the diffusion of a probe, NBD-labeled phosphatidylcholine, using fluorescence recovery after photobleaching. A transmembrane protein, the human delta-opioid receptor, was inserted into each lipid bilayer, and its ability to bind a synthetic ligand, DPDPE, cyclic[2-D-penicillamine, 5-D-penicillamine]enkephalin, was detected using single-molecule fluorescence spectroscopy by labeling this ligand with a rhodamine dye. The transmembrane protein was observed to bind the ligand for all bilayers tested. The protein's electrophoretic mobility was probed by monitoring the fluorescence from the bound ligand. The 5-nm polyacrylamide thickness gave the fastest diffusion for the fluorescent lipid probe (D 1 = 2.0(±1.2) × 10 -7 and D 2 = 1.2(±0.5) × 10 -6 cm 2/s) and also the largest electrophoretic mobility for the transmembrane protein (3 × 10 -8 cm 2/V·s). The optimum in polymer thickness is suggested to be a tradeoff between decoupling from the substrate and increasing roughness of the polymer surface.
ASJC Scopus subject areas
- Physical and Theoretical Chemistry
- Colloid and Surface Chemistry