Lipid bilayers on polyacrylamide brushes for inclusion of membrane proteins.
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.
Smith
Emily A
EA
Department of Chemistry, University of Arizona, Tucson, AZ 85721, USA.
Coym
Jason W
JW
Cowell
Scott M
SM
Tokimoto
Takahira
T
Hruby
Victor J
VJ
Yamamura
Henry I
HI
Wirth
Mary J
MJ
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
D000180:Acrylic Resins; D020881:Enkephalin, D-Penicillamine (2,5)-; D008024:Ligands; D008051:Lipid Bilayers; D008565:Membrane Proteins; D012822:Silicon Dioxide; D000099166:Polyacrylamides