title: Ultra-high vacuum surface analysis study of rhodopsin incorporation into supported lipid bilayers.
abstract: 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.
authors.affiliation: National ESCA and Surface Analysis Center for Biomedical Problems, Department of Bioengineering, Box 351750, University of Washington, Seattle, Washington 98195, USA.
authors.forename: Roger
authors.initials: R
authors.lastname: Michel
authors.forename: Varuni
authors.initials: V
authors.lastname: Subramaniam
authors.forename: Sally L
authors.initials: SL
authors.lastname: McArthur
authors.forename: Bruce
authors.initials: B
authors.lastname: Bondurant
authors.forename: Gemma D
authors.initials: GD
authors.lastname: D'Ambruoso
authors.forename: Henry K
authors.initials: HK
authors.lastname: Hall
authors.forename: Michael F
authors.initials: MF
authors.lastname: Brown
authors.forename: Eric E
authors.initials: EE
authors.lastname: Ross
authors.forename: S Scott
authors.initials: SS
authors.lastname: Saavedra
authors.forename: David G
authors.initials: DG
authors.lastname: Castner
mesh_terms: 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
chemical_list: D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D011108:Polymers; D002244:Carbon; D012243:Rhodopsin