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Insertion of Lipidated Ras Proteins into Lipid Monolayers Studied by Infrared Reflection Absorption Spectroscopy (IRRAS)

Annette Meister ^*, Chiara Nicolini , Herbert Waldmann , Jürgen Kuhlmann , Andreas Kerth ^*, Roland Winter  and Alfred Blume ^*

^* Institut für Physikalische Chemie, Martin-Luther-Universität Halle-Wittenberg, Halle, Germany; Fachbereich Chemie, Physikalische Chemie I – Biophysikalische Chemie, Universität Dortmund, Dortmund, Germany; and Abteilung für Chemische Biologie, and Abteilung für Strukturelle Biologie, Max-Planck-Institut für Molekulare Physiologie, Dortmund, Germany

Correspondence: Address reprint requests to Alfred Blume, Institute of Physical Chemistry, Martin-Luther-University Halle-Wittenberg, Muehlpforte 1, 06108 Halle/Saale, Germany. Tel.: 49-345-552-5850; Fax: 49-345-552-7157; E-mail: alfred.blume{at}chemie.uni-halle.de.

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.

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