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A novel method to calculate transition pathways between two known protein conformations is presented. It is based on a molecular dynamics simulation starting from one conformational state as initial structure and using the other for a directing constraint. The method is exemplified with the T ++ R transition of insulin. The most striking difference between these conformational states is that in T the 8 N-terminal residues of the B chain are arranged as an extended strand whereas in R they are forming a helix. Both the transition from T to R and from R to T were simulated. The method proves capable of finding a continuous pathway for each direction which are moderately different. The refolding processes are illustrated by a series of transient structures and pairs of a, t angles selected from the time course of the nimutations. In the T + R direction the helix is formed in the tast third of the transition, while in the R + T direction it is preserved during more than half of the simutation period. The results are discussed in comparison with those of an atternative method recently apptied to the T -. R transition of insulin which is based on targeted energy minimisation.

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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.

Protonated networks of internal water molecules appear to be involved in proton transfer in various integral membrane proteins. High-resolution x-ray studies of protein crystals at low temperature deliver mean positions of most internal waters, but only limited information about fluctuations within such H-bonded networks formed by water and residues. The question arises as to how water molecules behave inside and on the surface of a fluctuating membrane protein under more physiological conditions. Therefore, as an example, long-time molecular dynamics simulations of bacteriorhodopsin were performed with explicit membrane/water environment. Based on a recent x-ray model the bacteriorhodopsin trimer was inserted in a fully solvated 16 x 16 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)-bilayer patch, resulting in a system of approximately 84,000 atoms. Unrestrained molecular dynamics calculations of 5 ns were performed using the GROMACS package and force field. Mean water densities were computed to describe the anisotropic distribution of internal water molecules. In the whole protein two larger areas of higher water density are identified. They are located between the central proton binding site, the Schiff base, and the extracellular proton release site. Separated by Arg-82 these water clusters could provide a proton release pathway in a Grotthus-like mechanism as indicated by a continuum absorbance change observed during the photocycle by time-resolved Fourier transform infrared spectroscopy. Residues are identified which are H-bonded to the water clusters and are therefore facilitating proton conduction. Their influence on proton transfer via the H-bonded network as indicated by the continuum absorbance change is predicted. This may explain why several site-directed mutations alter the proton release kinetics without a direct involvement in proton transfer.

Members of the Ras superfamily regulate many cellular processes. They are down-regulated by a GTPase reaction in which GTP is cleaved into GDP and P i by nucleophilic attack of a water molecule. Ras proteins accelerate GTP hydrolysis by a factor of 10 5 compared to GTP in water. GTPase-activating proteins (GAPs) accelerate hydrolysis by another factor of 10 5 compared to Ras alone. Oncogenic mutations in Ras and GAPs slow GTP hydrolysis and are a factor in many cancers. Here, we elucidate in detail how this remarkable catalysis is brought about. We refined the protein-bound GTP structure and protein-induced charge shifts within GTP beyond the current resolution of X-ray structural models by combining quantum mechanics and molecular mechanics simulations with timeresolved Fourier-transform infrared spectroscopy. The simulations were validated by comparing experimental and theoretical IR difference spectra. The reactant structure of GTP is destabilized by Ras via a conformational change from a staggered to an eclipsed position of the nonbridging oxygen atoms of the γ-relative to the β-phosphates and the further rotation of the nonbridging oxygen atoms of α-relative to the β-and γ-phosphates by GAP. Further, the γ-phosphate becomes more positive although two of its oxygen atoms remain negative. This facilitates the nucleophilic attack by the water oxygen at the phosphate and proton transfer to the oxygen. Detailed changes in geometry and charge distribution in the ligand below the resolution of X-ray structure analysis are important for catalysis. Such high resolution appears crucial for the understanding of enzyme catalysis.enzyme catalysis | reaction mechanism | free energy of activation M any cellular processes are regulated by members of the Ras superfamily. They are switched "on" by GDP-to-GTP exchange and "off" by a GTPase reaction. GTP hydrolysis is vitally important for the regulation of several signal-transduction processes in living cells (1, 2). In the case of Ras, external growth signals are transduced to the nucleus. Site-specific oncogenic mutations inhibit the GTPase reaction and the growth signal can no longer be down-regulated. This contributes to uncontrolled cell growth, eventually leading to cancer (3). Common to all members of the Ras superfamily is the catalysis of GTP hydrolysis by the G domain (4). Ras-catalyzed GTP hydrolysis is five orders of magnitude faster than in water (30 min vs. 200 d) (5). However, to control growth signals in the living cell, a further increase of five orders of magnitude in the reaction rate (30 min to 50 ms) is enabled. This is effected by GTPase-activating proteins (GAPs) that interact with Ras (6). The mechanisms of GTP hydrolysis have been extensively investigated both theoretically (7-22) and experimentally by X-ray crystallography (23-27), electron spin resonance (28-30), and FTIR spectroscopy (5,6,(31)(32)(33).Determination of the three-dimensional structures of Ras and GAP by X-ray crystallography represents an important milestone in the understanding of t...

The GTPase Ras p21 is a crucial switch in cellular signal transduction. Fourier transform infrared (FTIR) spectra of the substrate guanosine triphosphate (GTP) show remarkable changes when it binds to the enzyme. The reduced band widths indicate that the flexible GTP molecule is guided by the protein into a preferred conformation. The delocalized phosphate vibrations of unbound GTP become localized. The frequency shifts show an electron movement toward beta-phosphate, which probably contributes to catalysis by reducing the free activation energy. To quantify these qualitative observations we performed QM/MM molecular dynamics simulations of Ras.GTP and GTP in water. The triphosphate part of GTP was treated quantum mechanically using density functional theory (DFT). Vibrational spectra were calculated in harmonic approximation with an average deviation of 3% from the experimental frequencies. This provides a high confidence in the computational results as vibrational spectra are highly sensitive to conformation and charge distribution. As compared to GTP in water, Ras-bound GTP shows a shift of negative charge of approximately 0.2 e toward the beta-phosphate from gamma-phosphate and from alpha-phosphate due to the positive charge of the magnesium ion, to a lesser extent of Lys-16, and surprisingly without any effect of the P-loop backbone. Magnesium and Gly-13 twist and bend the gamma-O-beta bonds such that the crucial bond is stretched before cleaving.

We here report on non-equilibrium targeted Molecular Dynamics simulations as tool for the estimation of protein-ligand unbinding kinetics. With this method, we furthermore investigate the molecular basis determining unbinding rates, correlating simulations with experimental data from SPR kinetics measurements and X-ray crystallography on two small molecule compound libraries bound to the N-terminal domain of the chaperone Hsp90. Within the investigated libraries, we find ligand conformational changes and protein-ligand nonbonded interactions as discriminators for unbinding rates. Ligands with flexible chemical scaffold may remain longer at the protein target if they need to pass through extended conformations upon unbinding, or if they exhibit strong electrostatic and/or van der Waals interactions with the target. Ligands with rigid chemical scaffold can exhibit longer residence times if they need to perform any kind of conformational change for unbinding, while electrostatic interactions with the protein can facilitate unbinding. Our resultsshow that understanding the unbinding pathway and the protein-ligand interactions along this path is crucial for the prediction of small molecule ligands with defined unbinding kinetics. Supporting InformationFour supporting tables, nine supporting figures and additional references (PDF) SMILES annotations (CSV) Accession CodesThe crystallographic coordinates of novel compounds are deposited in the Protein Data Bank under the accession codes 5LRL (2d) and 5LO1 (2j). Authors will release the atomic coordinates and experimental data upon article publication.