Ras is a central regulator of cellular signaling pathways. It is mutated in 20-30% of human tumors. To perform its function, Ras has to be bound to a membrane by a posttranslationally attached lipid anchor. Surprisingly, we identified here dimerization of membrane anchored Ras by combining attenuated total reflectance Fourier transform infrared spectroscopy, biomolecular simulations, and Förster resonance energy transfer experiments. By analyzing x-ray structural models and molecular-dynamics simulations, we propose a dimerization interface between α-helices 4 and 5 and the loop between β2 and β3. This seems to explain why the residues D47, E49, R135, R161, and R164 of this interface are influencing Ras signaling in cellular physiological experiments, although they are not positioned in the catalytic site. Dimerization could catalyze nanoclustering, which is well accepted for membrane-bound Ras. The interface could provide a new target for a seemingly novel type of small molecule interfering with signal transduction in oncogenic Ras mutants.

Due to the progress of density functional theory (DFT) accurate computations of vibrational spectra of isolated molecules have become a standard task in computational chemistry. This is not yet the case for solution spectra. To contribute to the exploration of corresponding computational procedures, here we suggest a more efficient variant of the so-called instantaneous normal-mode analysis (INMA). This variant applies conventional molecular dynamics (MD) simulations, which are based on nonpolarizable molecular mechanics (MM) force fields, to the rapid generation of a large ensemble of different solvation shells for a solute molecule. Short hybrid simulations, in which the solute is treated by DFT and the aqueous solvent by MM, start from snapshots of the MM solute−solvent MD trajectory and yield a set of statistically independent hydration shells partially adjusted to the DFT/MM force field. Within INMA, these shells are kept fixed at their 300 K structures, line spectra are calculated from the DFT/MM Hessians of the solute, and its inhomogeneously broadened solution spectra are derived by second-order statistics. As our test application we have selected the phosphate ions HPO4 2- and H2PO4 - because sizable solvation effects are expected for the IR spectra of these strongly polarizable ions. The widths, intensities, and spectral positions of the calculated bands are compared with experimental IR spectra recorded by us for the purpose of checking the computational procedures. These comparisons provide insights into the merits and limitations of the available DFT/MM approach to the prediction of IR spectra in the condensed phase.

Members of the Ras superfamily of small G proteins play key roles in signal transduction pathways, which they control by GTP hydrolysis. They are regulated by GTPase activating proteins (GAPs). Mutations that prevent hydrolysis cause severe diseases including cancer. A highly conserved ''arginine finger'' of GAP is a key residue. Here, we monitor the GTPase reaction of the Ras⅐RasGAP complex at high temporal and spatial resolution by time-resolved FTIR spectroscopy at 260 K. After triggering the reaction, we observe as the first step a movement of the switch-I region of Ras from the nonsignaling ''off'' to the signaling ''on'' state with a rate of 3 s ؊1 . The next step is the movement of the ''arginine finger'' into the active site of Ras with a rate of k2 ‫؍‬ 0.8 s ؊1 . Once the arginine points into the binding pocket, cleavage of GTP is fast and the protein-bound Pi intermediate forms. The switch-I reversal to the ''off'' state, the release of Pi, and the movement of arginine back into an aqueous environment is observed simultaneously with k3 ‫؍‬ 0.1 s ؊1 , the rate-limiting step. Arrhenius plots for the partial reactions show that the activation energy for the cleavage reaction is lowered by favorable positive activation entropy. This seems to indicate that protein-bound structured water molecules are pushed by the ''arginine finger'' movement out of the binding pocket into the bulk water. The proposed mechanism shows how the high activation barrier for phosphoryl transfer can be reduced by splitting into partial reactions separated by a Pi-intermediate.enzyme catalysis ͉ FTIR spectroscopy ͉ GTPases ͉ phosphate ͉ proteins

Targeted cancer therapies block cancer growth and spread using small molecules. Many molecular targets for an epidermal growth factor receptor (EGFR) selectively compete with the adenosine triphosphate-binding site of its tyrosine kinase domain. Detection of molecular targeted agents and their metabolites in cells/tissues by label-free imaging is attractive because dyes or fluorescent labels may be toxic or invasive. Here, label-free Raman microscopy is applied to show the spatial distribution of the molecular targeted drug erlotinib within the cell. The Raman images show that the drug is clustered at the EGFR protein at the membrane and induces receptor internalization. The changes within the Raman spectrum of erlotinib measured in cells as compared to the free-erlotinib spectrum indicate that erlotinib is metabolized within cells to its demethylated derivative. This study provides detailed insights into the drug targeting mechanism at the atomic level in cells. It demonstrates that Raman microscopy will open avenues as a non-invasive and label-free technique to investigate pharmacokinetics at the highest possible resolution in living cells.

The misfolding of the Amyloid-beta (Aβ) peptide into β-sheet enriched conformations was proposed as an early event in Alzheimer's Disease (AD). Here, the Aβ peptide secondary structure distribution in cerebrospinal fluid (CSF) and blood plasma of 141 patients was measured with an immuno-infrared-sensor. The sensor detected the amide I band, which reflects the overall secondary structure distribution of all Aβ peptides extracted from the body fluid. We observed a significant downshift of the amide I band frequency of Aβ peptides in Dementia Alzheimer type (DAT) patients, which indicated an overall shift to β-sheet. The secondary structure distribution of all Aβ peptides provides a better marker for DAT detection than a single Aβ misfold or the concentration of a specific oligomer. The discrimination between DAT and disease control patients according to the amide I frequency was in excellent agreement with the clinical diagnosis (accuracy 90% for CSF and 84% for blood). The amide I band maximum above or below the decisive marker frequency appears as a novel spectral biomarker candidate of AD. Additionally, a preliminary proof-of-concept study indicated an amide I band shift below the marker band already in patients with mild cognitive impairment due to AD. The presented immuno-IR-sensor method represents a promising, simple, robust, and label-free diagnostic tool for CSF and blood analysis.

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