Backbone Conformation and Dynamics of the Lipid-Modified Membrane Anchor of Human N-Ras Investigated by Solid-State NMR and Molecular Dynamics Simulations

Abstract

Many proteins involved in signal transduction are anchored to membranes by covalently attached lipid modifications. In this study we investigated the conformation and dynamics of the backbone and side chains of the N-Ras membrane anchoring domain. Experimental solid-state NMR studies involved doubly lipid-modified uniformly 13C and 15N labeled heptapeptides representing the C-terminus of N-Ras, which were incorporated into DMPC bilayers. A structural model of the peptide was calculated on the basis of isotropic chemical shifts, explicit torsion angle measurements, and nuclear Overhauser effects determined by solid-state NMR. The amplitude of molecular motions was assessed by 1H-13C order parameter measurements using separated local field NMR. For determination of the correlation times of the motions, T1 and T2 relaxation times were measured and analyzed using a generalized relaxation approach. To further understand the dynamics of Ras, molecular dynamics simulations of the molecule in lipid bilayers were conducted. In generating starting conditions for the simulation, special attention was paid to the backbone conformation since transitions between conformations were found to be rare events in a previous simulation of 100 ns length on this system [1]. Therefore, the experimentally determined conformation of the peptide backbone was equilibrated using a replica exchange technique in an explicit membrane environment. This enabled us to identify different conformers and to assess their relative probability. The resulting distribution of conformations was used subsequently for a long conventional MD simulation that was analyzed with regard to the experimental data. The combined simulations and experimental approach enabled a detailed model of the dynamics of the peptide to be obtained.[1] Vogel, A. Tan, K.-T. Waldmann, H. Feller, S.E. Brown, M.F. Huster, D. Biophys. J. 2007, 93, 2697-2712.