Free energy simulations for electrostatic and charging processes in complex molecular systems encounter specific difficulties owing to the long-range, 1/r Coulomb interaction. To calculate the solvation free energy of a simple ion, it is essential to take into account the polarization of nearby solvent but also the electrostatic potential drop across the liquid-gas boundary, however distant. The latter does not exist in a simulation model based on periodic boundary conditions because there is no physical boundary to the system. An important consequence is that the reference value of the electrostatic potential is not an ion in a vacuum. Also, in an infinite system, the electrostatic potential felt by a perturbing charge is conditionally convergent and dependent on the choice of computational conventions. Furthermore, with Ewald lattice summation and tinfoil conducting boundary conditions, the charges experience a spurious shift in the potential that depends on the details of the simulation system such as the volume fraction occupied by the solvent. All these issues can be handled with established computational protocols, as reviewed here and illustrated for several small ions and three solvated proteins.

Fatty acid photodecarboxylase (FAP) is a photoenzyme with potential green chemistry applications. By combining static, time-resolved, and cryotrapping spectroscopy and crystallography as well as computation, we characterized Chlorella variabilis FAP reaction intermediates on time scales from subpicoseconds to milliseconds. High-resolution crystal structures from synchrotron and free electron laser x-ray sources highlighted an unusual bent shape of the oxidized flavin chromophore. We demonstrate that decarboxylation occurs directly upon reduction of the excited flavin by the fatty acid substrate. Along with flavin reoxidation by the alkyl radical intermediate, a major fraction of the cleaved carbon dioxide unexpectedly transformed in 100 nanoseconds, most likely into bicarbonate. This reaction is orders of magnitude faster than in solution. Two strictly conserved residues, R451 and C432, are essential for substrate stabilization and functional charge transfer.

Tyrosine kinases transmit cellular signals through a complex mechanism, involving their phosphorylation and switching between inactive and active conformations. The cancer drug imatinib binds tightly to several homologous kinases, including Abl, but weakly to others, including Src. Imatinib specifically targets the inactive, so-called "DFG-out" conformation of Abl, which differs from the preferred, "DFG-in" conformation of Src in the orientation of a conserved Asp-Phe-Gly (DFG) activation loop. However, recent x-ray structures showed that Src can also adopt the DFG-out conformation and uses it to bind imatinib. The Src/Abl-binding free energy difference can thus be decomposed into two contributions. Contribution i measures the different protein-imatinib interactions when either kinase is in its DFG-out conformation. Contribution ii depends on the ability of imatinib to select or induce this conformation, i.e. on the relative stabilities of the DFG-out and DFG-in conformations of each kinase. Neither contribution has been measured experimentally. We use molecular dynamics simulations to show that contribution i is very small, 0.2 ؎ 0.6 kcal/mol; imatinib interactions are very similar in the two kinases, including long range electrostatic interactions with the imatinib positive charge. Contribution ii, deduced using the experimental binding free energy difference, is much larger, 4.4 ؎ 0.9 kcal/mol. Thus, conformational selection, easy in Abl, difficult in Src, underpins imatinib specificity. Contribution ii has a simple interpretation; it closely approximates the stability difference between the DFG-out and DFG-in conformations of apo-Src. Additional calculations show that conformational selection also governs the relative binding of imatinib to the kinases c-Kit and Lck. These results should help clarify the current framework for engineering kinase signaling.

More than 100 naturally occurring modified nucleotides have been found in RNA molecules, in particular in tRNAs. We have determined molecular mechanics force field parameters compatible with the CHARMM36 all-atom additive force field for all these modifications using the CHARMM force field parametrization strategy. Emphasis was placed on fine tuning of the partial atomic charges and torsion angle parameters. Quantum mechanics calculations on model compounds provided the initial set of target data, and extensive molecular dynamics simulations of nucleotides and oligonucleotides in aqueous solutions were used for further refinement against experimental data. The presented parameters will allow for computational studies of wide range of RNAs containing modified nucleotides including the ribosome and transfer RNAs.

Resistance to the antibiotic tetracycline (Tc) is regulated by its binding as a Tc:Mg2+ complex to the Tet Repressor protein (TetR). Tc:TetR recognition is a complex problem, with the protein and ligand each having several possible conformations and protonation states, which are difficult to elucidate by experiment alone. We used a combination of free-energy simulations and crystallographic analysis to investigate the electrostatic interactions between protein and ligand and the possible role of induced fit in Tc binding. Tc in solution was described quantum mechanically, while Tc:TetR interactions were described by a recent, high-quality molecular-mechanics model. The orientations of the amide and imidazole groups were determined experimentally by a careful analysis of Debye-Waller factors in alternate crystallographic models. The agreement with experiment for these orientations suggested that the simulations and their more detailed, thermodynamic predictions were reliable. We found that the ligand prefers an extended, zwitterionic state both in solution and in complexation with the protein. Tc is thus preorganized for binding, while the protein combines lock-and-key behavior for regions close to the ligand's amide, enolate, and ammonium groups, with an induced fit for regions close to the Mg2+ ion. These insights and the modeling techniques employed should be of interest for engineering improved TetR ligands and improved TetR proteins for gene regulation, as well as for drug design.

The acid/base properties of proteins are essential in biochemistry, and proton binding is a valuable reporter on electrostatic interactions. We propose a constant-pH Monte Carlo strategy to compute protonation free energies and pK(a)'s. The solvent is described implicitly, through a generalized Born model. The electronic polarizability and backbone motions of the protein are included through the protein dielectric constant. Side chain motions are described explicitly, by the Monte Carlo scheme. An efficient computational algorithm is described, which allows us to treat the fluctuating shape of the protein/solvent boundary in a way that is numerically exact (within the GB framework); this contrasts with several previous constant-pH approaches. For a test set of six proteins and 78 titratable groups, the model performs well, with an rms error of 1.2 pH units. While this is slightly greater than a simple Null model (rms error of 1.1) and a fully empirical model (rms error of 0.9), it is obtained using physically meaningful model parameters, including a low protein dielectric of four. Importantly, similar performance is obtained for side chains with large and small pK(a) shifts (relative to a standard model compound). The titration curve slopes and the conformations sampled are reasonable. Several directions to improve the method further are discussed.

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Most of the factors involved in translation (tRNA, rRNA and proteins) are subject to post-transcriptional and post-translational modifications, which participate in the fine-tuning and tight control of ribosome and protein synthesis processes. In eukaryotes, Trm112 acts as an obligate activating platform for at least four methyltransferases (MTase) involved in the modification of 18S rRNA (Bud23), tRNA (Trm9 and Trm11) and translation termination factor eRF1 (Mtq2). Trm112 is then at a nexus between ribosome synthesis and function. Here, we present a structure-function analysis of the Trm9-Trm112 complex, which is involved in the 5-methoxycarbonylmethyluridine (mcm5U) modification of the tRNA anticodon wobble position and hence promotes translational fidelity. We also compare the known crystal structures of various Trm112-MTase complexes, highlighting the structural plasticity allowing Trm112 to interact through a very similar mode with its MTase partners, although those share less than 20% sequence identity.