Markus Meuwly

Allosteric control of cyclic di-GMP signaling

Cyclic di-guanosine monophosphate is a bacterial second messenger that has been implicated in biofilm formation, antibiotic resistance, and persistence of pathogenic bacteria in their animal host. Although the enzymes responsible for the regulation of cellular levels of c-di-GMP, diguanylate cyclases (DGC) and phosphodiesterases, have been identified recently, little information is available on the molecular mechanisms involved in controlling the activity of these key enzymes or on the specific interactions of c-di-GMP with effector proteins. By using a combination of genetic, biochemical, and modeling techniques we demonstrate that an allosteric binding site for c-di-GMP (I-site) is responsible for non-competitive product inhibition of DGCs. The I-site was mapped in both multi- and single domain DGC proteins and is fully contained within the GGDEF domain itself. In vivo selection experiments and kinetic analysis of the evolved I-site mutants led to the definition of an RXXD motif as the core c-di-GMP binding site. Based on these results and based on the observation that the I-site is conserved in a majority of known and potential DGC proteins, we propose that product inhibition of DGCs is of fundamental importance for c-di-GMP signaling and cellular homeostasis. The definition of the I-site binding pocket provides an entry point into unraveling the molecular mechanisms of ligand-protein interactions involved in c-di-GMP signaling and makes DGCs a valuable target for drug design to develop new strategies against biofilm-related diseases.

Study of the insulin dimerization : Binding free energy calculations and per-residue free energy decomposition

A calculation of the binding free energy for the dimerization of insulin has been performed using the molecular mechanics–generalized Born surface area approach. The calculated absolute binding free energy is −11.9 kcal/mol, in approximate agreement with the experimental value of −7.2 kcal/mol. The results show that the dimerization is mainly due to nonpolar interactions. The role of the hydrogen bonds between the 2 monomers appears to give the direction of the interactions. A per‐atom decomposition of the binding free energy has been performed to identify the residues contributing most to the self association free energy. Residues B24–B26 are found to make the largest favorable contributions to the dimerization. Other residues situated at the interface between the 2 monomers were found to make favorable but smaller contributions to the dimerization: Tyr B16, Val B12, and Pro B28, and to an even lesser extent, Gly B23. The energy decomposition on a per‐residue basis is in agreement with experimental alanine scanning data. The results obtained from a single trajectory (i.e., the dimer trajectory is also used for the monomer analysis) and 2 trajectories (i.e., separate trajectories are used for the monomer and dimer) are similar. Proteins 2005.

MORPHING AB INITIO POTENTIALS : A SYSTEMATIC STUDY OF NE-HF

A procedure for “morphing” an ab initio potential energy surface to obtain agreement with experimental data is presented. The method involves scaling functions for both the energy and the intermolecular distance. In the present work, the scaling functions are parametrized and determined by least-squares fitting to the experimental data. The method is tested on the system Ne–HF, for which high-resolution infrared spectra are available. It is shown to work well even with relatively low-level ab initio calculations. Several basis sets are investigated at the CCSD(T) correlation level, including various aug-cc-pVnZ basis sets and the specially-tailored Ne–HF basis set of ONeil et al. All give good results after morphing, but the changes needed to match experiment are much smaller for the ONeil basis set. The use of MP2 calculations is also investigated: again, the MP2 potential is quite satisfactory after morphing, but requires much more modification than the CCSD(T) potential.

Theoretical Investigation of Infrared Spectra and Pocket Dynamics of Photodissociated Carbonmonoxy Myoglobin

Molecular dynamics simulations of the photodissociated state of carbonmonoxy myoglobin (MbCO) are presented using a fluctuating charge model for CO. A new three-point charge model is fitted to high-level ab initio calculations of the dipole and quadrupole moment functions taken from the literature. The infrared spectrum of the CO molecule in the heme pocket is calculated using the dipole moment time autocorrelation function and shows good agreement with experiment. In particular, the new model reproduces the experimentally observed splitting of the CO absorption spectrum. The splitting of 3-7 cm(-1) (compared to the experimental value of 10 cm(-1)) can be directly attributed to the two possible orientations of CO within the docking site at the edge of the distal heme pocket (the B states), as previously suggested on the basis of experimental femtosecond time-resolved infrared studies. Further information on the time evolution of the position and orientation of the CO molecule is obtained and analyzed. The calculated difference in the free energy between the two possible orientations (Fe...CO and Fe...OC) is 0.3 kcal mol(-1) and agrees well with the experimentally estimated value of 0.29 kcal mol(-1). A comparison of the new fluctuating charge model with an established fixed charge model reveals some differences that may be critical for the correct prediction of the infrared spectrum and energy barriers. The photodissociation of CO from the myoglobin mutant L29F using the new model shows rapid escape of CO from the distal heme pocket, in good agreement with recent experimental data. The effect of the protein environment on the multipole moments of the CO ligand is investigated and taken into account in a refined model. Molecular dynamics simulations with this refined model are in agreement with the calculations based on the gas-phase model. However, it is demonstrated that even small changes in the electrostatics of CO alter the details of the dynamics.

Double proton transfer in the isolated and DNA-embedded guanine-cytosine base pair.

The energetics and dynamics of double proton transfer (DPT) is investigated theoretically for the Watson-Crick conformation of the guanine-cytosine (GC) base pair. Using semiempirical density functional theory the isolated and DNA-embedded GC pair is considered. Differences in the energetics and dynamics of DPT thus addresses the question of how relevant studies of isolated base pairs are for the understanding of processes occurring in DNA. Two-dimensional potential energy surfaces involving the transferring hydrogen atoms and the proton donors and acceptors are presented for both systems. The DPT reaction is accompanied by a contraction of the distance between the two bases with virtually identical energetic barriers being 18.8 and 18.7 kcal/mol for the isolated and DNA-embedded system, respectively. However, the transition state for DPT in the DNA-embedded GC pair is offset by 0.1 A to larger N-H separation compared to the isolated GC pair. Using activated ab initio molecular dynamics, DPT is readily observed for the isolated base pair with a minimal amount of 21.4 kcal/mol of initial average kinetic energy along the DPT normal mode vector. On a time scale of approximately 100 fs DPT has occurred and the excess energy is redistributed. For the DNA-embedded GC pair considerably more kinetic energy is required (30.0 kcal/mol) for DPT and the process is completed within one hydrogen vibration. The relevance of studies of isolated base pairs and base pair analogs in regard of reactions or properties involving DNA is discussed.

Atomistic Simulation of Adiabatic Reactive Processes Based on Multi-State Potential Energy Surfaces.

The adiabatic reactive molecular dynamics (ARMD) method provides a framework to study chemical reactions using molecular dynamics simulations with minimal computational overhead. Here, ARMD is generalized to an arbitrary reactive process between two states in which reactants and products can be treated by an atomistic force field. The implementation is described, and the method is applied to two systems: the kinetics of NO rebinding to myoglobin (Mb) as a validation system and the conformational transition in neuroglobin (Ngb) which explores the full functionality of ARMD. For MbNO, the nonexponential kinetics observed both in experiment and earlier ARMD studies is reproduced. Furthermore, the sensitivity of the results with respect to the asymptotic separation between the two potential energy surfaces (NO bound and unbound) is studied.

The role of higher CO-multipole moments in understanding the dynamics of photodissociated carbonmonoxide in myoglobin.

The influence of electrostatic multipole moments up to hexadecapole on the dynamics of photodissociated carbon monoxide (CO) in myoglobin is investigated. The CO electrostatic potential is expressed as an expansion into atomic multipole moments of increasing order up to octopole which are obtained from a distributed multipole analysis. Three models with increasingly accurate molecular multipoles (accurate quadrupole, octopole, and hexadecapole moments, respectively) are developed and used in molecular dynamics simulations. All models with a fluctuating quadrupole moment correctly describe the location of the B-state whereas the sign of the octopole moment differentiates between the Fe...CO and Fe...OC orientation. For the infrared spectrum of photodissociated CO, considerable differences between the three electrostatic models are found. The most detailed electrostatic model correctly reproduces the splitting, shift, and width of the CO spectrum in the B-state. From an analysis of the trajectories, the spectroscopic B(1) and B(2) states are assigned to the Fe...CO and Fe...OC substates, respectively.

Simulation of proton transfer along ammonia wires: An “ab initio” and semiempirical density functional comparison of potentials and classical molecular dynamics

Protonated ammonia clusters of the composition (NxH3x+1)+ with x=2,3,4 are investigated by using the gradient corrected, three-parameter functional by Becke based on the functional by Lee, Yang, and Parr (B3LYP/6-31G**) and self-consistent charges density functional tight-binding (SCC–DFTB) methods for calculating the potential energy surface and forces in the Born–Oppenheimer approximation. They are used for classical molecular dynamics simulations at temperatures ranging from 5 K to 600 K. Results from the two methods are compared for proton transfer in N2H7+. The number of proton transfer events as a function of temperature is similar, although at low temperatures, SCC–DFTB cuts off more rapidly than B3LYP/6-31G**. Calculated vibrational spectra agree well for the intermolecular N–N and intramolecular N–H stretch excitations. Both approaches lead to broad, relatively unstructured bands extending over about 1500 cm−1 for the proton transfer coordinate. Simulations at the SCC–DFTB/MD level for larger (Nx...

Mid‐infrared spectra of the proton‐bound complexes Nen–HCO+ (n=1,2)

The ν1 band of Ne–HCO+ has been recorded for both 20Ne and 22Ne containing isotopomers by means of infrared photodissociation spectroscopy. The rotational structure of the band is consistent with a parallel Σ–Σ type transition of a linear proton‐bound complex. The following constants are extracted for 20Ne–HCO+: ν0=3046.120±0.006 cm−1, B″=0.099 54±0.000 05 cm−1, D″=(5.30±0.30)×10−7 cm−1, H″=(1.1±0.9)×10−11 cm−1, B′=0.100 03±0.000 05 cm−1, D′=(4.89±0.30)×10−7 cm−1, H′=(1.6±0.9)×10−11 cm−1. The ν1 band is redshifted by 42.5 cm−1 from the corresponding ν1 transition of free HCO+ indicating that the Ne atom has a pronounced influence on the proton motion. Linewidths for individual rovibrational transitions are laser bandwidth limited, demonstrating that the lifetime of the ν1 level is at least 250 ps. An approximate radial potential for the collinear Ne...HCO+ interaction is constructed by joining the mid‐range potential obtained from a Rydberg–Klein–Rees inversion of the spectroscopic data to the theoretical...

Mid‐infrared spectra of He–HN+2 and He2–HN+2

Mid‐infrared vibrational spectra of He–HN+ 2 and He2–HN+ 2 have been recorded by monitoring their photofragmentation in a tandem mass spectrometer. For He–HN+ 2 three rotationally resolved bands are seen: the fundamental ν1 transition (N–H stretch) at 3158.419±0.009 cm−1, the ν1+ν b combination band (N–H stretch plus intermolecular bend) at 3254.671±0.050 cm−1, and the ν1+ν s combination band (N–H stretch plus intermolecular stretch) at 3321.466±0.050 cm−1. The spectroscopic data facilitate the development of approximate one‐dimensional radial intermolecular potentials relevant to the collinear bonding of He to HN+ 2 in its (000) and (100) vibrational states. These consist of a short range potential derived from an RKR inversion of the spectroscopic data, together with a long range polarization potential generated by considering the interaction between the He atom and a set of multipoles distributed on the HN+ 2 nuclei. The following estimates for binding energies are obtained: D 0 ″=378 cm−1 [He+HN+ 2(000)], and D 0 ′=431 cm−1 [He+HN+ 2(100)]. While the ν1 band of He2–HN+ 2 is not rotationally resolved, the fact that it is barely shifted from the corresponding band of He–HN+ 2 suggests that the trimer possesses a structure in which one of the He atoms occupies a linear proton‐bound position forming a He–HN+ 2 core, to which a second less strongly bound He is attached.