Insertion of lipidated Ras proteins into lipid monolayers studied by infrared reflection absorption spectroscopy (IRRAS).
Ras proteins have to be associated with the inner leaflet of the plasma membrane to perform their signaling functions. This membrane targeting and binding is controlled by post-translational covalent attachment of farnesyl and palmitoyl chains to cysteines in the membrane anchor region of the N- and H-Ras isoforms. Two N-Ras lipoproteins were investigated, namely a farnesylated and hexadecylated protein, presenting the natural hydrophobic modifications and a doubly hexadecylated construct, respectively. The proteins are surface active and form a Gibbs monolayer at the air-D2O interface. The contours of the amide-I bands were analyzed using infrared reflection absorption spectroscopy (IRRAS). Langmuir monolayers of a mixture of POPC, brain sphingomyelin, and cholesterol were used as half of a model biomembrane to study the insertion of these N-Ras proteins. They insert with their hydrophobic anchors into lipid monolayers but at higher surface pressures (30 mN/m); the farnesylated and hexadecylated protein desorbs completely from the monolayer, whereas the doubly hexadecylated protein remains incorporated. During the insertion process, changes in the orientation of the protein secondary structure were detected by comparison with simulated IRRA spectra, based on the information on the relative orientation of the secondary structure elements from the protein crystal structure data.
Meister
Annette
A
Institut für Physikalische Chemie, Martin-Luther-Universität Halle-Wittenberg, Halle, Germany.
Nicolini
Chiara
C
Waldmann
Herbert
H
Kuhlmann
Jürgen
J
Kerth
Andreas
A
Winter
Roland
R
Blume
Alfred
A
D000327:Adsorption; D001665:Binding Sites; D003198:Computer Simulation; D008051:Lipid Bilayers / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry*; D008956:Models, Chemical; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D013055:Spectrophotometry, Infrared; D018631:ras Proteins / Q000032:analysis / Q000737:chemistry*
D008051:Lipid Bilayers; D008565:Membrane Proteins; D018631:ras Proteins