Lipid modifications of a Ras peptide exhibit altered packing and mobility versus host membrane as detected by 2H solid-state NMR.

The human N-ras protein binds to cellular membranes by insertion of two covalently bound posttranslational lipid modifications, which is crucial for its function in signal transduction and cell proliferation. Mutations in ras may lead to unregulated cell growth and eventually cancer, making it an important therapeutic target. Here we have investigated the molecular details of the membrane binding mechanism. A heptapeptide derived from the C-terminus of the human N-ras protein was synthesized including two hexadecyl modifications. Solid-state 2H NMR was used to determine the packing and molecular dynamics of the ras lipid chains as well as the phospholipid matrix. Separately labeling the chains of the peptide and the phospholipids with 2H enabled us to obtain atomically resolved parameters relevant to their structural dynamics. While the presence of ras only marginally affected the packing of DMPC membranes, dramatically lower order parameters (S(CD)) were observed for the ras acyl chains indicating modified packing properties. Essentially identical projected lengths of the 16:0 ras chains and the 14:0 DMPC chains were found, implying that the polypeptide backbone is located at the lipid-water interface. Dynamical properties of both the ras and phospholipid chains were determined from spin-lattice 2H relaxation (R1Z) measurements. Plots of R1Z rates versus the corresponding squared segmental order parameters revealed striking differences. We propose the ras peptide is confined to microdomains containing DMPC chains which are in exchange with the bulk bilayer on the 2H NMR time scale (approximately 10(-5) s). Compared to the host DMPC matrix, the ras lipid modifications are extremely flexible and undergo relatively large amplitude motions. It is hypothesized that this flexibility is a requirement for the optimal anchoring of lipid-modified proteins to cellular membranes.

Traceless Fischer indole synthesis on the solid phase.

The Fischer indole synthesis using polymer-bound hydrazines is employed as the key step for the development of a traceless indole synthesis on a solid support.

Membrane insertion of a lipidated ras peptide studied by FTIR, solid-state NMR, and neutron diffraction spectroscopy.

Membrane binding of a doubly lipid modified heptapeptide from the C-terminus of the human N-ras protein was studied by Fourier transform infrared, solid-state NMR, and neutron diffraction spectroscopy. The 16:0 peptide chains insert well into the 1,2-dimyristoyl-sn-glycero-3-phosphocholine phospholipid matrix. This is indicated by a common main phase transition temperature of 21.5 degrees C for both the lipid and peptide chains as revealed by FTIR measurements. Further, (2)H NMR reveals that peptide and lipid chains have approximately the same chain length in the liquid crystalline state. This is achieved by a much lower order parameter of the 16:0 peptide chains compared to the 14:0 phospholipid chains. Finally, proton/deuterium contrast variation of neutron diffraction experiments indicates that peptide chains are localized in the membrane interior analogous to the phospholipid chains. In agreement with this model of peptide chain insertion, the peptide part is localized at the lipid-water interface of the membrane. This is revealed by (1)H nuclear Overhauser enhancement spectra recorded under magic angle spinning conditions. Quantitative cross-peak analysis allows the examination of the average location of the peptide backbone and side chains with respect to the membrane. While the backbone shows the strongest cross-relaxation rates with the phospholipid glycerol, the hydrophobic side chains of the peptide insert deeper into the membrane interior. This is supported by neutron diffraction experiments that reveal a peptide distribution in the lipid-water interface of the membrane. Concurring with these experimental findings, the amide protons of the peptide show strong water exchange as seen in NMR and FTIR measurements. No indications for a hydrogen-bonded secondary structure of the peptide backbone are found. Therefore, membrane binding of the C-terminus of the N-ras protein is mainly due to lipid chain insertion but also supported by interactions between hydrophobic side chains and the lipid membrane. The peptide assumes a mobile and disordered conformation in the membrane. Since the C-terminus of the soluble part of the ras protein is also disordered, we hypothesize that our model for membrane binding of the ras peptide realistically describes the membrane binding of the lipidated C-terminus of the active ras protein.