Ultra-high vacuum surface analysis study of rhodopsin incorporation into supported lipid bilayers.
Planar supported lipid bilayers that are stable under ambient atmospheric and ultra-high-vacuum conditions were prepared by cross-linking polymerization of bis-sorbylphosphatidylcholine (bis-SorbPC). X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) were employed to investigate bilayers that were cross-linked using either redox-initiated radical polymerization or ultraviolet photopolymerization. The redox method yields a more structurally intact bilayer; however, the UV method is more compatible with incorporation of transmembrane proteins. UV polymerization was therefore used to prepare cross-linked bilayers with incorporated bovine rhodopsin, a light-activated, G-protein-coupled receptor (GPCR). A previous study (Subramaniam, V.; Alves, I. D.; Salgado, G. F. J.; Lau, P. W.; Wysocki, R. J.; Salamon, Z.; Tollin, G.; Hruby, V. J.; Brown, M. F.; Saavedra, S. S. J. Am. Chem. Soc. 2005, 127, 5320-5321) showed that rhodopsin retains photoactivity after incorporation into UV-polymerized bis-SorbPC, but did not address how the protein is associated with the bilayer. In this study, we show that rhodopsin is retained in supported bilayers of poly(bis-SorbPC) under ultra-high-vacuum conditions, on the basis of the increase in the XPS nitrogen concentration and the presence of characteristic amino acid peaks in the ToF-SIMS data. Angle-resolved XPS data show that the protein is inserted into the bilayer, rather than adsorbed on the bilayer surface. This is the first study to demonstrate the use of ultra-high-vacuum techniques for structural studies of supported proteolipid bilayers.
Michel
Roger
R
National ESCA and Surface Analysis Center for Biomedical Problems, Department of Bioengineering, Box 351750, University of Washington, Seattle, Washington 98195, USA.
Subramaniam
Varuni
V
McArthur
Sally L
SL
Bondurant
Bruce
B
D'Ambruoso
Gemma D
GD
Hall
Henry K
HK
Brown
Michael F
MF
Ross
Eric E
EE
Saavedra
S Scott
SS
Castner
David G
DG
D000818:Animals; D002244:Carbon / Q000737:chemistry; D002417:Cattle; D008051:Lipid Bilayers / Q000737:chemistry*; D013058:Mass Spectrometry; D010084:Oxidation-Reduction; D010713:Phosphatidylcholines / Q000737:chemistry*; D011108:Polymers / Q000737:chemistry; D012243:Rhodopsin / Q000737:chemistry*; D013053:Spectrophotometry; D013499:Surface Properties; D014618:Vacuum
D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D011108:Polymers; D002244:Carbon; D012243:Rhodopsin