Residual structure in a peptide fragment of the outer membrane protein X under denaturing conditions: a molecular dynamics study.

The Escherichia coli outer membrane protein X (OmpX) contains two polypeptide segments that present nonrandom residual structure in 8 M aqueous urea, whereas the remainder of the protein is in a flexibly disordered conformation (Tafer et al. in Biochemistry 43:860-869, 2004). In the present study, the results of two long-timescale (0.4 micros) unrestrained explicit-solvent molecular dynamics (MD) simulations of a tetradecapeptide representative of one of these two segments in 8 M aqueous urea are reported and analyzed. The two simulations were initiated either from the conformation of the corresponding segment in an NMR model structure of the unfolded protein or from an entirely extended configuration. The sampled conformational ensembles agree qualitatively with the experimentally observed NOEs, but not quantitatively, suggesting that a number of relevant configurations were not visited on the 2 x 0.4 micros timescale. Major conformational transitions occur on the 0.1 micros timescale, and the ensembles corresponding to the two independent simulations overlap only to a limited extent. However, both simulations show in multiple events the reversible formation and disruption of alpha-helical secondary structure (characteristic of the urea-denatured state) and beta-turn secondary structure (characteristic of the native state). Events of helix formation are correlated with the appearance of hydrogen bonds between two side chains (Asp75-Ser78) and of a persistent hydrophobic contact (Trp76-Tyr80). They also evidence a peculiar helix stabilization and N-terminal capping role for a negatively charged residue (Asp75). These features are in good qualitative agreement with the NMR model for the structured state of the corresponding segment in the urea-denatured protein. The analysis of the simulations provides a detailed picture of the structural and dynamic features of the considered peptide at atomic resolution that is of high relevance in the understanding of the OmpX folding process.

Molecular dynamics simulations of a reversibly folding beta-heptapeptide in methanol: influence of the treatment of long-range electrostatic interactions.

Eight 100-ns molecular dynamics simulations of a beta-heptapeptide in methanol at 340 K (within cubic periodic computational boxes of about 6-nm edge) are reported and compared. These simulations were performed with three different charge-state combinations at the peptide termini, one of them with or without a neutralizing chloride counterion, and using either the lattice-sum (LS) or reaction-field (RF) scheme to handle electrostatic interactions. The choice of the electrostatic scheme has essentially no influence on the folding-unfolding equilibrium when the peptide termini are uncharged and only a small influence when the peptide is positively charged at its N-terminus (with or without inclusion of a neutralizing chloride counterion). However, when the peptide is zwitterionic, the LS scheme leads to preferential sampling of the high-dipole folded helical state, whereas the RF scheme leads to preferential sampling of a low-dipole unfolded salt-bridged state. A continuum electrostatics analysis based on the sampled configurations (zwitterionic case) suggests that the LS scheme stabilizes the helical state through artificial periodicity, but that the magnitude of this perturbation is essentially negligible (compared to the thermal energy) for the large box size and relatively polar solvent considered. The results thus provide clear evidence (continuum electrostatics analysis) for the absence of LS artifacts and some indications (still not definitive because of the limited sampling of the folding-unfolding transition) for the presence of RF artifacts in this specific system.

Conformation, dynamics, solvation and relative stabilities of selected beta-hexopyranoses in water: a molecular dynamics study with the GROMOS 45A4 force field.

The present article reports long timescale (200 ns) simulations of four beta-D-hexopyranoses (beta-D-glucose, beta-D-mannose, beta-D-galactose and beta-D-talose) using explicit-solvent (water) molecular dynamics and vacuum stochastic dynamics simulations together with the GROMOS 45A4 force field. Free-energy and solvation free-energy differences between the four compounds are also calculated using thermodynamic integration. Along with previous experimental findings, the present results suggest that the formation of intramolecular hydrogen-bonds in water is an 'opportunistic' consequence of the close proximity of hydrogen-bonding groups, rather than a major conformational driving force promoting this proximity. In particular, the conformational preferences of the hydroxymethyl group in aqueous environment appear to be dominated by 1,3-syn-diaxial repulsion, with gauche and solvation effects being secondary, and intramolecular hydrogen-bonding essentially negligible. The rotational dynamics of the exocyclic hydroxyl groups, which cannot be probed experimentally, is found to be rapid (10-100 ps timescale) and correlated (flip-flop hydrogen-bonds interconverting preferentially through an asynchronous disrotatory pathway). Structured solvent environments are observed between the ring and lactol oxygen atoms, as well as between the 4-OH and hydroxymethyl groups. The calculated stability differences between the four compounds are dominated by intramolecular effects, while the corresponding differences in solvation free energies are small. An inversion of the stereochemistry at either C(2) or C(4) from equatorial to axial is associated with a raise in free energy. Finally, the particularly low hydrophilicity of beta-D-talose appears to be caused by the formation of a high-occurrence hydrogen-bonded bridge between the 1,3-syn-diaxial 2-OH and 4-OH groups. Overall, good agreement is found with available experimental and theoretical data on the structural, dynamical, solvation and energetic properties of these compounds. However, this detailed comparison also reveals some discrepancies, suggesting the need (and providing a solid basis) for further refinement.

A multiple time step algorithm compatible with a large number of distance classes and an arbitrary distance dependence of the time step size for the fast evaluation of nonbonded interactions in molecular simulations.

A new algorithm is introduced to perform the multiple time step integration of the equations of motion for a molecular system, based on the splitting of the nonbonded interactions into a series of distance classes. The interactions between particle pairs in successive classes are updated at a progressively decreasing frequency. Unlike previous multiple time-stepping schemes relying on distance classes, the present algorithm sorts interacting particle pairs by their next update times rather than by their update frequencies. For this reason, the proposed scheme is extremely flexible with respect to the number of classes that can be employed (up to hundred or more) and the distance dependence of the relative time step size (arbitrary integer function of the distance). It can also easily be adapted to classes defined based on a criterion other than the interparticle distance (e.g., interaction magnitude). Different variants of the algorithm are tested in terms of accuracy and efficiency for simulations of a pure water system (6167 molecules) under truncated-octahedral periodic boundary conditions, and compared to the twin-range method standardly used with GROMOS96 (short- and long-range cutoff distances of 0.8 and 1.4 nm, pair list and intermediate-range interactions updated every five steps). In particular, multiple time-stepping schemes with an accuracy comparable to that of the twin-range method can be designed, that permit to increase the effective (long-range) cutoff distance from 1.4 to 3.0 nm with a performance loss of only about a factor 2. This result is quite encouraging, considering the benefits of doubling the cutoff radius in the context of (bio-)molecular simulations.

The GROMOS software for biomolecular simulation: GROMOS05.

We present the latest version of the Groningen Molecular Simulation program package, GROMOS05. It has been developed for the dynamical modelling of (bio)molecules using the methods of molecular dynamics, stochastic dynamics, and energy minimization. An overview of GROMOS05 is given, highlighting features not present in the last major release, GROMOS96. The organization of the program package is outlined and the included analysis package GROMOS++ is described. Finally, some applications illustrating the various available functionalities are presented.

Use of molecular dynamics in the design and structure determination of a photoinducible beta-hairpin.

The study presented here consists of three parts. In the first, the ability of a set of differently substituted diazobenzene-based linkers to act as photoswitchable beta-turn building blocks was assessed. A 12-residue peptide known to form beta-hairpins was taken as the basis for the modeling process. The central (beta-turn) residue pair was successively replaced by six symmetrically ((o,o), (m,m), or (p,p)) substituted (aminomethyl/carboxymethyl or aminoethyl/carboxyethyl) diazobenzene derivatives leading to a set of peptides with a photoswitchable backbone conformation. The folding behavior of each peptide was then investigated by performing molecular dynamics simulations in water (4 ns) and in methanol (10 ns) at room temperature. The simulations suggest that (o,o)- and (m,m)-substituted linkers with a single methylene spacer are significantly better suited to act as photoswitchable beta-turn building blocks than the other linkers examined in this study. The peptide containing the (m,m)-substituted linker was synthesized and characterized by NMR in its cis configuration. In the second part of this study, the structure of this peptide was refined using explicit-solvent simulations and NOE distance restraints, employing a variety of refinement protocols (instantaneous and time-averaged restraining as well as unrestrained simulations). We show that for this type of systems, even short simulations provide a significant improvement in our understanding of their structure if physically meaningful force fields are employed. In the third part, unrestrained explicit-solvent simulations starting from either the NMR model structure (75 ns) or a fully extended structure (25 ns) are shown to converge to a stable beta-hairpin. The resulting ensemble is in good agreement with experimental data, indicating successful structure prediction of the investigated hairpin by classical explicit-solvent molecular dynamics simulations.

A photoinducible beta-hairpin.

A photochromic azobenzene linker was incorporated as a turn element into an amino acid sequence known to fold into a beta-hairpin structure in aqueous solution. Oligomer formation when the linker was in its thermodynamically favored trans form prohibited structure determination. Light-induced conformational change of the linker to the cis form led to the formation of monomers which exhibited a well-defined beta-hairpin structure as determined by (1)H NMR. The rate of the light-induced cis-to-trans isomerization of the azobenzene-containing peptide was 30% slower compared to the unsubstituted chromophore. These results suggest that suitably substituted azobenzenes can be used as photoinducible turn elements to investigate and control the folding and stability of beta-sheets.

An improved nucleic acid parameter set for the GROMOS force field.

Over the past decades, the GROMOS force field for biomolecular simulation has primarily been developed for performing molecular dynamics (MD) simulations of polypeptides and, to a lesser extent, sugars. When applied to DNA, the 43A1 and 45A3 parameter sets of the years 1996 and 2001 produced rather flexible double-helical structures, in which the Watson-Crick hydrogen-bonding content was more limited than expected. To improve on the currently available parameter sets, the nucleotide backbone torsional-angle parameters and the charge distribution of the nucleotide bases are reconsidered based on quantum-chemical data. The new 45A4 parameter set resulting from this refinement appears to perform well in terms of reproducing solution NMR data and canonical hydrogen bonding. The deviation between simulated and experimental observables is now of the same order of magnitude as the uncertainty in the experimental values themselves.