Visualizing association of N-ras in lipid microdomains: influence of domain structure and interfacial adsorption.
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.
Nicolini
Chiara
C
Physical Chemistry I-Biophysical Chemistry and Organic Chemistry, Department of Chemistry, University of Dortmund, Otto-Hahn-Strasse 6, D-44227 Dortmund, Germany.
Baranski
Jörg
J
Schlummer
Stefanie
S
Palomo
José
J
Lumbierres-Burgues
Maria
M
Kahms
Martin
M
Kuhlmann
Jürgen
J
Sanchez
Susana
S
Gratton
Enrico
E
Waldmann
Herbert
H
Winter
Roland
R
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
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