QM/MM Study of L-Lactate Oxidation by Flavocytochrome b2

In this work, we have performed molecular dynamics simulations using a hybrid Quantum Mechanics/Molecular Mechanics (QM/MM) scheme to study the mechanism of L-lactate oxidation by flavocytochrome b2 (Fcb2). Our results obtained at the QM(AM1)/MM level have been improved by single-point corrections using density functional theory (DFT) methods. Free energy surfaces have been calculated in the framework of the hydride transfer hypothesis. This mechanism involves the transfer of the lactate hydroxyl proton to H373 while the substrate αH atom is transferred as a hydride to the flavin mononucleotide (FMN) prosthetic group anchored in the active site. Four different systems have been modeled: wild-type enzyme considering R289 in a distal or a proximal conformation observed in crystal structures and the D282N and Y254L variants (with R289 in a distal position). Simulation results highlight the influence of the environment on the catalytic mechanism by describing a step-wise process in the WT enzyme with R289 in a distal position and a concerted mechanism for the other systems. In the step-wise mechanism, the hydride transfer to flavin can occur only after a proton transfer from substrate to H373. Modifications of the electrostatic field around L-lactate or H373 disfavor the highly charged complex resulting from this proton transfer. Simulations of the Y254L variant also reveal some effect of steric changes

Poor adherence to antibiotic prescribing guidelines in acute otitis media—obstacles, implications, and possible solutions

Many countries now have guidelines on the clinical management of acute otitis media. In almost all, the public health goal of containing acquired resistance in bacteria through reduced antibiotic prescribing is the main aim and basis for recommendations. Despite some partial short-term successes, clinical activity databases and opinion surveys suggest that such restrictive guidelines are not followed closely, so this aim is not achieved. Radical new solutions are needed to tackle irrationalities in healthcare systems which set the short-term physician–patient relationship against long-term public health. Resolving this opposition will require comprehensive policy appraisal and co-ordinated actions at many levels, not just dissemination of evidence and promotion of guidelines. The inappropriate clinical rationales that underpin non-compliance with guidelines can be questioned by evidence, but also need specific developments promoting alternative solutions, within a framework of whole-system thinking. Promising developments would be (a) physician training modules on age-appropriate analgesia and on detection plus referral of rare complications like mastoiditis, and (b) vaccination against the most common and serious bacterial pathogens

Using biased molecular dynamics and Brownian dynamics in the study of fluorescent proteins

cited By 7International audienceThe present article presents a theoretical study of the dynamics of the chromophore of the Green Fluorescent Protein in its excited state on a long time scale (a few ten nanoseconds) in order to help the interpretation of time resolved experiments. The starting hypothesis is that the quenching of fluorescence is related to the internal motion of the chromophore, usually called 'φ torsion'. In fact, that motion is hindered by the protein and cannot be studied by standard molecular dynamics. Therefore we have developed a different approach involving three steps. First the potential of mean force (PMF) along the considered torsion is obtained by biased molecular dynamics (umbrella sampling). Then, a long time scale, single particle Brownian dynamics is performed using that PMF and an appropriate diffusion constant. Finally we determine the nth passage time (NPT) distribution functions at geometries or regions where nonradiative ground state recovery may occur. The NPT distributions generalize the more usual 'mean first passage time' and allow determining different quantities like fluorescence decay profiles, mean fluorescence lifetime, quantum yield etc. These quantities are used here in a qualitative discussion of the fluorescence decay in Green Fluorescent Protein

Relation between pH, structure, and absorption spectrum of Cerulean: A study by molecular dynamics and TD DFT calculations

cited By 6International audienceMolecular dynamics (MD) and quantum mechanical calculations of the Cerulean green fluorescent protein (a variant of enhanced cyan fluorescent protein ECFP) at pH 5.0 and 8.0 are presented, addressing two questions arising from experimental results (Malo et al., Biochemistry 2007;46:9865 - 9873): the origin of the blue shift of absorption spectrum when the pH is decreased from 8.0 to 5.0, and the lateral chain orientation of the key residue Asp 148. We demonstrate that the blue shift is reproduced assuming that a rotation around the single bond of the exocydie ring of the chromophore takes place when the pH changes from 5.0 to 8.0. We find that Asp 148 is protonated and inside the barrel at pH 5.0 in agreement with crystallographic data. However, the hydrogen bond pattern of Asp 148 is different in simulations of the solvated protein and in the crystal structure. This difference is explained by a partial closing of the cleft between strands 6 and 7 in MD simulations. This study provides also a structure at pH 8.0: the Asp 148 carboxylate group is exposed to the solvent and the chromophore is stabilized in the trans conformation by a tighter hydrogen bond network. This work gives some insight into the relationship between the pH and the chromophore conformation and suggests an interpretation of the very similar fluorescent properties of ECFP and ECFP/ H148D

Excited state dynamics of the green fluorescent protein on the nanosecond time scale

cited By 18International audienceWe have introduced a new algorithm in the parallel processing PMEMD module of the AMBER suite that allows MD simulations with a potential involving two coupled torsions. We have used this modified module to study the green fluorescent protein. A coupled torsional potential was adjusted on high accuracy quantum chemical calculations of the anionic chromophore in the first excited state, and several 15-ns-long MD simulations were performed. We have obtained an estimate of the fluorescence lifetime (2.2 ns) to be compared to the experimental value (3 ns), which is, to the best of our knowledge, the first theoretical estimate of that lifetime

Cyan fluorescent protein: Molecular dynamics, simulations, and electronic absorption spectrum

cited By 30International audienceThe dynamics and electronic absorption spectrum of enhanced cyan fluorescent protein (ECFP), a mutant of green fluorescent protein (GFP), have been studied by means of a 1 ns molecular dynamics (MD) simulation. The two X-ray conformations A′ and B′ of ECFP were considered. The chromophore was assumed to be neutral, and all titratable residues were taken in their standard protonation state at neutral pH. The protein was embedded in a box of water molecules (and counterions). The first result is that the two conformations A′ and B′ are found to be stable all along the simulation. Then, an analysis of the hydrogen-bond networks shows strong differences between the two conformations in the surroundings of the nitrogen atom of the indolic part of the chromophore. This is partly due to the imperfection in the β barrel near the His148 residue, which allows the access of one solvent molecule inside the protein in conformation A′. Finally, quantum mechanical calculations of the electronic transition energies of the chromophore in the charge cloud of the protein and solvent water molecules were performed using the TDDFT method on 160 snapshots extracted every 5 ps of the MD trajectories. It is found that conformations A′ and B′ exhibit very similar spectra despite different H-bond networks involving the chromophore. This similarity is related to the weak charge transfer involved in the electronic transition and the weak electrostatic field created by ECFP near the chromophore, within the hypotheses made in the present simulation

Molecular simulation studies of water physisorption in zeolites.

International audienc