Calcium Inhibition of Ribonuclease H1 Two-Metal Ion Catalysis

Most phosphate-processing enzymes require Mg2+ as a cofactor to catalyze nucleotide cleavage and transfer reactions. Ca2+ ions inhibit many of these enzymatic activities, despite Ca2+ and Mg2+ having comparable binding affinities and overall biological abundances. Here we study the molecular details of the calcium inhibition mechanism for phosphodiester cleavage, an essential reaction in the metabolism of nucleic acids and nucleotides, by comparing Ca2+- and Mg2+ catalyzed reactions. We study the functional roles of the specific metal ion sites A and B in enabling the catalytic cleavage of an RNA/DNA hybrid substrate by B. halodurans ribonuclease (RNase) H1 using hybrid quantum-mechanics/molecular mechanics (QM/MM) free energy calculations. We find that Ca2+ substitution of either of the two active-site Mg2+ ions substantially increases the height of the reaction barrier and thereby abolishes the catalytic activity. Remarkably, Ca2+ at the A site is inactive also in Mg2+-optimized active-site structures along the reaction path, whereas Mg2+ substitution recovers activity in Ca2+-optimized structures. Geometric changes resulting from Ca2+ substitution at metal ion site A may thus be a secondary factor in the loss of catalytic activity. By contrast, at metal ion site B geometry plays a more important role, with only a partial recovery of activity after Mg2+substitution in Ca2+-optimized structures. Ca2+-substitution also leads to a change in mechanism, with deprotonation of the water nucleophile requiring a closer approach to the scissile phosphate, which in turn increases the barrier. As a result, Ca2+ is less efficient in activating the water. As a likely cause for the different reactivities of Mg2+ and Ca2+ ions in site A, we identify differences in charge transfer to the ions and the associated decrease in the pKa of the oxygen nucleophile attacking the phosphate group

Efficient Irreversible Monte Carlo samplers

We present here two irreversible Markov chain Monte Carlo algorithms for general discrete state systems, one of the algorithms is based on the random-scan Gibbs sampler for discrete states and the other on its improved version, the Metropolized-Gibbs sampler. The algorithms we present incorporate the lifting framework with skewed detailed balance condition and construct irreversible Markov chains that satisfy the balance condition. We have applied our algorithms to 1D 4-state Potts model. The integrated autocorrelation times for magnetisation and energy density indicate a reduction of the dynamical scaling exponent from $z \approx 1$ to $z \approx 1/2$. In addition, we have generalized an irreversible Metropolis-Hastings algorithm with skewed detailed balance, initially introduced by Turitsyn et al. (2011) for the mean field Ising model, to be now readily applicable to classical spin systems in general; application to 1D 4-state Potts model indicate a square root reduction of the mixing time at high temperatures.Comment: The document consists of 45 pages and 32 figure

Cucurbit[8]uril-mediated pseudo[2,3]rotaxanes.

Pseudo[2,3]rotaxanes have been successfully fabricated by the complexation of cucurbit[8]uril (CB[8]) macrocycles with extended viologen derivatives. Two design rules enable the incorporation of a third CB[8] onto a recently reported pseudo[2,2]rotaxane. Incorporation of a third macrocycle confines the dimeric stacking of chromophores into specific alignment, leading to effective electron-delocalisation along their long molecular axis.- Leverhulme Trust (project: “Natural material innovation for sustainable living”), - EPSRC Programme Grant (NOtCH, EP/L027151/1), - ERC-2016 Consolidator Grant (CAM-RIG, 726470,), - EPSRC (EP/R013012/1, EP/N020669/1), - BBSRC (BB/N007700/1), - ERC Starting Grant (BioNet,757850

Simulated tempering with irreversible Gibbs sampling techniques

We present here two novel algorithms for simulated tempering simulations, which break detailed balance condition (DBC) but satisfy the skewed detailed balance to ensure invariance of the target distribution. The irreversible methods we present here are based on Gibbs sampling and concern breaking DBC at the update scheme of the temperature swaps. We utilise three systems as a test bed for our methods: an MCMC simulation on a simple system described by a 1D double well potential, the Ising model and MD simulations on Alanine pentapeptide (ALA5). The relaxation times of inverse temperature, magnetic susceptibility and energy density for the Ising model indicate clear gains in sampling efficiency over conventional Gibbs sampling techniques with DBC and also over the conventionally used simulated tempering with Metropolis-Hastings (MH) scheme. Simulations on ALA5 with large number of temperatures indicate distinct gains in mixing times for inverse temperature and consequently the energy of the system compared to conventional MH. With no additional computational overhead, our methods were found to be more efficient alternatives to conventionally used simulated tempering methods with DBC. Our algorithms should be particularly advantageous in simulations of large systems with many temperature ladders, as our algorithms showed a more favorable constant scaling in Ising spin systems as compared with both reversible and irreversible MH algorithms. In future applications, our irreversible methods can also be easily tailored to utilize a given dynamical variable other than temperature to flatten rugged free energy landscapes

Molecular Vibration Explorer: an Online Database and Toolbox for Surface-Enhanced Frequency Conversion and Infrared and Raman Spectroscopy

We present Molecular Vibration Explorer, a freely accessible online database and interactive tool for exploring vibrational spectra and tensorial light-vibration coupling strengths of a large collection of thiolated molecules. The "Gold" version of the database gathers the results from density functional theory calculations on 2800 commercially available thiol compounds linked to a gold atom, with the main motivation to screen the best molecules for THz and mid-infrared to visible upconversion. Additionally, the "Thiol" version of the database contains results for 1900 unbound thiolated compounds. They both provide access to a comprehensive set of computed spectroscopic parameters for all vibrational modes of all molecules in the database. The user can simultaneously investigate infrared absorption, Raman scattering, and vibrational sum- and difference-frequency generation cross sections. Molecules can be screened for various parameters in custom frequency ranges, such as a large Raman cross-section under a specific molecular orientation, or a large orientation-averaged sum-frequency generation (SFG) efficiency. The user can select polarization vectors for the electromagnetic fields, set the orientation of the molecule, and customize parameters for plotting the corresponding IR, Raman, and sum-frequency spectra. We illustrate the capabilities of this tool with selected applications in the field of surface-enhanced spectroscopy

Representation of the QM Subsystem for Long-Range Electrostatic Interaction in Non-Periodic Ab Initio QM/MM Calculations

This paper is published as part of a thematic issue of Molecules on “Combined Quantum Mechanical and Molecular Mechanical Methods and Simulations”. http://www.mdpi.com/journal/molecules/special_issues/QMIn QM/MM calculations, it is essential to handle electrostatic interactions between the QM and MM subsystems accurately and efficiently. To achieve maximal efficiency, it is convenient to adopt a hybrid scheme, where the QM electron density is used explicitly in the evaluation of short-range QM/MM electrostatic interactions, while a multipolar representation for the QM electron density is employed to account for the long-range QM/MM electrostatic interactions. In order to avoid energy discontinuity at the cutoffs, which separate the short- and long-range QM/MM electrostatic interactions, a switching function should be utilized to ensure a smooth potential energy surface. In this study, we benchmarked the accuracy of such hybrid embedding schemes for QM/MM electrostatic interactions using different multipolar representations, switching functions and cutoff distances. For test systems (neutral and anionic oxyluciferin in MM (aqueous and enzyme) environments), the best accuracy was acquired with a combination of QM electrostatic potential (ESP) charges and dipoles and two switching functions (long-range electrostatic corrections (LREC) and Switch) in the treatment of long-range QM/MM electrostatics. It allowed us to apply a 10Å distance cutoff and still obtain QM/MM electrostatics/polarization energies within 0.1 kcal/mol and time-dependent density functional theory (TDDFT)/MM vertical excitation energies within 10−3 eV from theoretical reference values.This research was funded by the U.S. Department of Energy Office of Science (DE-SC0011297), EPSRC (EP/R013012/1, EP/L027151/1 and EP/N020669), and ERC (Project 757850 BioNet).Ye

Mechanism-Based Redesign of GAP to Activate Oncogenic Ras

Ras GTPases play a crucial role in cell signaling pathways. Mutations of the Ras gene occur in about one third of cancerous cell lines and are often associated with detrimental clinical prognosis. Hot spot residues Gly12, Gly13, and Gln61 cover 97% of oncogenic mutations, which impair the enzymatic activity in Ras. Using QM/MM free energy calculations, we present a two-step mechanism for the GTP hydrolysis catalyzed by the wild-type Ras.GAP complex. We found that the deprotonation of the catalytic water takes place via the Gln61 as a transient Brønsted base. We also determined the reaction profiles for key oncogenic Ras mutants G12D and G12C using QM/MM minimizations, matching the experimentally observed loss of catalytic activity, thereby validating our reaction mechanism. Using the optimized reaction paths, we devised a fast and accurate procedure to design GAP mutants that activate G12D Ras. We replaced GAP residues near the active site and determined the activation barrier for 190 single mutants. We furthermore built a machine learning for ultrafast screening, by fast prediction of the barrier heights, tested both on the single and double mutations. This work demonstrates that fast and accurate screening can be accomplished via QM/MM reaction path optimizations to design protein sequences with increased catalytic activity. Several GAP mutations are predicted to re-enable catalysis in oncogenic G12D, offering a promising avenue to overcome aberrant Ras-driven signal transduction by activating enzymatic activity instead of inhibition. The outlined computational screening protocol is readily applicable for designing ligands and cofactors analogously

Insights into functions of the H channel of cytochrome c oxidase from atomistic molecular dynamics simulations

Proton pumping A-type cytochrome c oxidase (CcO) terminates the respiratory chains of mitochondria and many bacteria. Three possible proton transfer pathways (D, K, and H channels) have been identified based on structural, functional, and mutational data. Whereas the D channel provides the route for all pumped protons in bacterial A-type CcOs, studies of bovine mitochondrial CcO have led to suggestions that its H channel instead provides this route. Here, we have studied H-channel function by performing atomistic molecular dynamics simulations on the entire, as well as core, structure of bovine CcO in a lipid-solvent environment. The majority of residues in the H channel do not undergo large conformational fluctuations. Its upper and middle regions have adequate hydration and H-bonding residues to form potential proton-conducting channels, and Asp51 exhibits conformational fluctuations that have been observed crystallographically. In contrast, throughout the simulations, we do not observe transient water networks that could support proton transfer from the N phase toward heme a via neutral His413, regardless of a labile H bond between Ser382 and the hydroxyethylfarnesyl group of heme a. In fact, the region around His413 only became sufficiently hydrated when His413 was fixed in its protonated imidazolium state, but its calculated pK(a) is too low for this to provide the means to create a proton transfer pathway. Our simulations show that the electric dipole moment of residues around heme a changes with the redox state, hence suggesting that the H channel could play a more general role as a dielectric well.Peer reviewe