title: Visualizing association of N-ras in lipid microdomains: influence of domain structure and interfacial adsorption.
abstract: In this study, two-photon fluorescence microscopy on giant unilamellar vesicles and tapping-mode atomic force microscopy (AFM) are applied to follow the insertion of a fluorescently (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene, BODIPY) labeled and completely lipidated (hexadecylated and farnesylated) N-Ras protein into heterogeneous lipid bilayer systems. The bilayers consist of the canonical raft mixture 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), sphingomyelin, and cholesterol, which-depending on the concentration of the constituents-separates into liquid-disordered (l(d)), liquid-ordered (l(o)), and solid-ordered (s(o)) phases. The results provide direct evidence that partitioning of N-Ras occurs preferentially into liquid-disordered lipid domains, which is also reflected in a faster kinetics of incorporation into the fluid lipid bilayers. The phase sequence of preferential binding of N-Ras to mixed-domain lipid vesicles is l(d) > l(o) >> s(o). Intriguingly, we detect, using the better spatial resolution of AFM, also a large proportion of the lipidated protein located at the l(d)/l(o) phase boundary, thus leading to a favorable decrease in line tension that is associated with the rim of the demixed phases. Such an interfacial adsorption effect may serve as an alternative vehicle for association processes of signaling proteins in membranes.
authors.affiliation: Physical Chemistry I-Biophysical Chemistry and Organic Chemistry, Department of Chemistry, University of Dortmund, Otto-Hahn-Strasse 6, D-44227 Dortmund, Germany.
authors.forename: Chiara
authors.initials: C
authors.lastname: Nicolini
authors.forename: Jörg
authors.initials: J
authors.lastname: Baranski
authors.forename: Stefanie
authors.initials: S
authors.lastname: Schlummer
authors.forename: José
authors.initials: J
authors.lastname: Palomo
authors.forename: Maria
authors.initials: M
authors.lastname: Lumbierres-Burgues
authors.forename: Martin
authors.initials: M
authors.lastname: Kahms
authors.forename: Jürgen
authors.initials: J
authors.lastname: Kuhlmann
authors.forename: Susana
authors.initials: S
authors.lastname: Sanchez
authors.forename: Enrico
authors.initials: E
authors.lastname: Gratton
authors.forename: Herbert
authors.initials: H
authors.lastname: Waldmann
authors.forename: Roland
authors.initials: R
authors.lastname: Winter
mesh_terms: D001896:Boron Compounds / Q000737:chemistry; D002784:Cholesterol / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D008563:Membrane Lipids / Q000737:chemistry* / Q000378:metabolism; D021962:Membrane Microdomains / Q000737:chemistry* / Q000378:metabolism; D018625:Microscopy, Atomic Force; D008856:Microscopy, Fluorescence / Q000379:methods; D008958:Models, Molecular; D010713:Phosphatidylcholines / Q000737:chemistry; D013109:Sphingomyelins / Q000737:chemistry; D018631:ras Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D005456:Fluorescent Dyes; D008563:Membrane Lipids; D010713:Phosphatidylcholines; D013109:Sphingomyelins; D002784:Cholesterol; D018631:ras Proteins; C028694:1-palmitoyl-2-oleoylphosphatidylcholine