title: Lipid bilayers on polyacrylamide brushes for inclusion of membrane proteins.
abstract: 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) x 10(-7) and D(2) = 1.2(+/-0.5) x 10(-6) cm(2)/s) and also the largest electrophoretic mobility for the transmembrane protein (3 x 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.
authors.affiliation: Department of Chemistry, University of Arizona, Tucson, AZ 85721, USA.
authors.forename: Emily A
authors.initials: EA
authors.lastname: Smith
authors.forename: Jason W
authors.initials: JW
authors.lastname: Coym
authors.forename: Scott M
authors.initials: SM
authors.lastname: Cowell
authors.forename: Takahira
authors.initials: T
authors.lastname: Tokimoto
authors.forename: Victor J
authors.initials: VJ
authors.lastname: Hruby
authors.forename: Henry I
authors.initials: HI
authors.lastname: Yamamura
authors.forename: Mary J
authors.initials: MJ
authors.lastname: Wirth
mesh_terms: D000180:Acrylic Resins; D020881:Enkephalin, D-Penicillamine (2,5)- / Q000032:analysis / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D008051:Lipid Bilayers / Q000032:analysis* / Q000737:chemistry; D008565:Membrane Proteins / Q000032:analysis / Q000737:chemistry*; D012822:Silicon Dioxide; D013050:Spectrometry, Fluorescence; D000099166:Polyacrylamides
chemical_list: D000180:Acrylic Resins; D020881:Enkephalin, D-Penicillamine (2,5)-; D008024:Ligands; D008051:Lipid Bilayers; D008565:Membrane Proteins; D012822:Silicon Dioxide; D000099166:Polyacrylamides