Visualizing Association of N-Ras in Lipid Microdomains:  Influence of Domain Structure and Interfacial Adsorption

• Published in: Journal of the American Chemical Society Vol. 128; no. 1; pp. 192 - 201
• Main Authors: Nicolini, Chiara, Baranski, Jörg, Schlummer, Stefanie, Palomo, José, Lumbierres-Burgues, Maria, Kahms, Martin, Kuhlmann, Jürgen, Sanchez, Susana, Gratton, Enrico, Waldmann, Herbert, Winter, Roland
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 11.01.2006
• Subjects: Biological and medical sciences; Boron Compounds - chemistry; Cholesterol - chemistry; Fluorescent Dyes - chemistry; Fundamental and applied biological sciences. Psychology; Membrane Lipids - chemistry; Membrane Lipids - metabolism; Membrane Microdomains - chemistry; Membrane Microdomains - metabolism; Microscopy, Atomic Force; Microscopy, Fluorescence - methods; Models, Molecular; Molecular biophysics; Phosphatidylcholines - chemistry; ras Proteins - chemistry; ras Proteins - metabolism; Sphingomyelins - chemistry; Surface properties. Adsorption; Atomic force microscopy; Two photon excitation; Fluorescent tracer; Organic adsorbate; Phospholipid; Biomembrane; Tricyclic compound; Organic adsorbent; Organic solvent; Liquid liquid interface; Liquid liquid adsorption; Adsorption structure; Nitrogen boron heterocycle; Fluorescence microscopy
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja055779x

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 (ld), liquid-ordered (lo), and solid-ordered (so) 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 ld > lo ≫ so. Intriguingly, we detect, using the better spatial resolution of AFM, also a large proportion of the lipidated protein located at the ld/lo 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.