Loop-loop interaction in an adenine-sensing riboswitch: a molecular dynamics study.

Riboswitches are mRNA-based molecules capable of controlling the expression of genes. They undergo conformational changes upon ligand binding, and as a result, they inhibit or promote the expression of the associated gene. The close connection between structural rearrangement and function makes a detailed knowledge of the molecular interactions an important step to understand the riboswitch mechanism and efficiency. We have performed all-atom molecular dynamics simulations of the adenine-sensing add A-riboswitch to study the breaking of the kissing loop, one key tertiary element in the aptamer structure. We investigated the aptamer domain of the add A-riboswitch in complex with its cognate ligand and in the absence of the ligand. The opening of the hairpins was simulated using umbrella sampling using the distance between two loops as the reaction coordinate. A two-step process was observed in all the simulated systems. First, a general loss of stacking and hydrogen bond interactions is seen. The last interactions that break are the two base pairs G37-C61 and G38-C60, but the break does not affect the energy profile, indicating their pivotal role in the tertiary structure formation but not in the structure stabilization. The junction area is partially organized before the kissing loop formation and residue A24 anchors together the loop helices. Moreover, when the distance between the loops is increased, one of the hairpins showed more flexibility by changing its orientation in the structure, while the other conserved its coaxial arrangement with the rest of the structure.

Nucleotide modifications and tRNA anticodon-mRNA codon interactions on the ribosome.

We have carried out molecular dynamics simulations of the tRNA anticodon and mRNA codon, inside the ribosome, to study the effect of the common tRNA modifications cmo(5)U34 and m(6)A37. In tRNA(Val), these modifications allow all four nucleotides to be successfully read at the wobble position in a codon. Previous data suggest that entropic effects are mainly responsible for the extended reading capabilities, but detailed mechanisms have remained unknown. We have performed a wide range of simulations to elucidate the details of these mechanisms at the atomic level and quantify their effects: extensive free energy perturbation coupled with umbrella sampling, entropy calculations of tRNA (free and bound to the ribosome), and thorough structural analysis of the ribosomal decoding center. No prestructuring effect on the tRNA anticodon stem-loop from the two modifications could be observed, but we identified two mechanisms that may contribute to the expanded decoding capability by the modifications: The further reach of the cmo(5)U34 allows an alternative outer conformation to be formed for the noncognate base pairs, and the modification results in increased contacts between tRNA, mRNA, and the ribosome.

Direct epoxidation in Candida antarctica lipase B studied by experiment and theory.

Candida antarctica lipase B (CALB) is a promiscuous serine hydrolase that, besides its native function, catalyzes different side reactions, such as direct epoxidation. A single-point mutant of CALB demonstrated a direct epoxidation reaction mechanism for the epoxidation of alpha,beta-unsaturated aldehydes by hydrogen peroxide in aqueous and organic solution. Mutation of the catalytically active Ser105 to alanine made the previously assumed indirect epoxidation reaction mechanism impossible. Gibbs free energies, activation parameters, and substrate selectivities were determined both computationally and experimentally. The energetics and mechanism for the direct epoxidation in CALB Ser105Ala were investigated by density functional theory calculations, and it was demonstrated that the reaction proceeds through a two step-mechanism with formation of an oxyanionic intermediate. The active-site residue His224 functions as a general acid-base catalyst with support from Asp187. Oxyanion stabilization is facilitated by two hydrogen bonds from Thr40.

Motions and entropies in proteins as seen in NMR relaxation experiments and molecular dynamics simulations.

Molecular dynamics simulations of E. coli glutaredoxin1 in water have been performed to relate the dynamical parameters and entropy obtained in NMR relaxation experiments, with results extracted from simulated trajectory data. NMR relaxation is the most widely used experimental method to obtain data on dynamics of proteins, but it is limited to relatively short timescales and to motions of backbone amides or in some cases (13)C-H vectors. By relating the experimental data to the all-atom picture obtained in molecular dynamics simulations, valuable insights on the interpretation of the experiment can be gained. We have estimated the internal dynamics and their timescales by calculating the generalized order parameters (O) for different time windows. We then calculate the quasiharmonic entropy (S) and compare it to the entropy calculated from the NMR-derived generalized order parameter of the amide vectors. Special emphasis is put on characterizing dynamics that are not expressed through the motions of the amide group. The NMR and MD methods suffer from complementary limitations, with NMR being restricted to local vectors and dynamics on a timescale determined by the rotational diffusion of the solute, while in simulations, it may be difficult to obtain sufficient sampling to ensure convergence of the results. We also evaluate the amount of sampling obtained with molecular dynamics simulations and how it is affected by the length of individual simulations, by clustering of the sampled conformations. We find that two structural turns act as hinges, allowing the α helix between them to undergo large, long timescale motions that cannot be detected in the time window of the NMR dipolar relaxation experiments. We also show that the entropy obtained from the amide vector does not account for correlated motions of adjacent residues. Finally, we show that the sampling in a total of 100 ns molecular dynamics simulation can be increased by around 50%, by dividing the trajectory into 10 replicas with different starting velocities.

Magnesium Ion-Water Coordination and Exchange in Biomolecular Simulations.

Magnesium ions have an important role in the structure and folding mechanism of ribonucleic acid systems. To properly simulate these biophysical processes, the applied molecular models should reproduce, among other things, the kinetic properties of the ions in water solution. Here, we have studied the kinetics of the binding of magnesium ions with water molecules and nucleic acid systems using molecular dynamics simulation in detail. We have validated the parameters used in biomolecular force fields, such as AMBER and CHARMM, for Mg(2+) ions and also for the biologically relevant ions Na(+), K(+), and Ca(2+) together with three different water models (TIP3P, SPC/E, and TIP5P). The results show that Mg(2+) ions have a slower exchange rate than Na(+), K(+), and Ca(2+) in agreement with the experimental trend, but the simulated value underestimates the experimentally observed Mg(2+)-water exchange rate by several orders of magnitude, irrespective of the force field and water model. A new set of parameters for Mg(2+) was developed to reproduce the experimental kinetic data. This set also leads to better reproduction of structural data than existing models. We have applied the new parameter set to Mg(2+) binding with a monophosphate model system and with the purine riboswitch, add A-riboswitch. In line with the Mg(2+)-water results, the newly developed parameters show a better description of the structure and kinetics of the Mg(2+)-phosphate binding than all other models. The characterization of the ion binding to the riboswitch system shows that the new parameter set does not affect the global structure of the ribonucleic acid system or the number of ions involved in direct or indirect binding. A slight decrease in the number of water-bridged contacts between A-riboswitch and the Mg(2+) ion is observed. The results support the ability of the newly developed parameters to improve the kinetic description of the Mg(2+) and phosphate ions and their applicability in nucleic acid simulation.