Ultra-High Vacuum Surface Analysis Study of Rhodopsin Incorporation into Supported Lipid Bilayers

• Published in: Langmuir Vol. 24; no. 9; pp. 4901 - 4906
• Main Authors: Michel, Roger, Subramaniam, Varuni, McArthur, Sally L, Bondurant, Bruce, D'Ambruoso, Gemma D, Hall, Henry K, Brown, Michael F, Ross, Eric E, Saavedra, S. Scott, Castner, David G
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 06.05.2008
• Subjects: Animals; Carbon - chemistry; Cattle; Chemistry; Colloidal state and disperse state; Exact sciences and technology; General and physical chemistry; Lipid Bilayers - chemistry; Mass Spectrometry; Oxidation-Reduction; Phosphatidylcholines - chemistry; Polymers - chemistry; Rhodopsin - chemistry; Spectrophotometry; Surface physical chemistry; Surface Properties; Vacuum; Incorporation; Bovine; Support; Polymerization; Lipids; Secondary ion mass spectrometry; X ray; Surface analysis; Nitrogen; Bilayer; Protein; Vertebrata; Mammalia; Aminoacid; Photoelectron spectrometry; Artiodactyla; Ungulata
• ISSN: 0743-7463; 1520-5827
• DOI: 10.1021/la800037r

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.