Optimization of the CHARMM additive force field for DNA: Improved treatment of the BI/BII conformational equilibrium

The B-form of DNA can populate two different backbone conformations: BI and BII, defined by the difference between the torsion angles ε and ζ (BI = ε-ζ < 0 and BII = ε-ζ > 0). BI is the most populated state, but the population of the BII state, which is sequence dependent, is significant and accumulating evidence shows that BII affects the overall structure of DNA, and thus influences protein-DNA recognition. This work presents a reparametrization of the CHARMM27 additive nucleic acid force field to increase the sampling of the BII form in MD simulations of DNA. In addition, minor modifications of sugar puckering were introduced to facilitate sampling of the A form of DNA under the appropriate environmental conditions. Parameter optimization was guided by quantum mechanical data on model compounds, followed by calculations on several DNA duplexes in the condensed phase. The selected optimized parameters were then validated against a number of DNA duplexes, with the most extensive tests performed on the EcoRI dodecamer, including comparative calculations using the Amber Parm99bsc0 force field. The new CHARMM model better reproduces experimentally observed sampling of the BII conformation, including sampling as a function of sequence. In addition, the model reproduces the A form of the 1ZF1 duplex in 75 % ethanol, and yields a stable Z-DNA conformation of duplex (GTACGTAC) in its crystal environment. The resulting model, in combination with a recent reoptimization of the CHARMM27 force field for RNA, will be referred to as CHARMM36.     

Conformations and dynamics of the phosphodiester backbone of a DNA fragment that bears a strong topoisomerase II cleavage site

The dynamics of the DNA phosphodiester backbone conformations have been studied for a strong topoisomerase II cleavage site (site 22) using molecular dynamics simulations in explicit water and in the presence of sodium ions. We investigated the backbone motions and more particularly the BI/BII transitions involving the epsilon and zeta angles. The consensus cleavage site is adjacent to the phosphate which shows the most important phosphodiester backbone flexibility in the sequence. We infer that these latter properties could be responsible for the preferential cleavage at this site possibly through the perturbation of the cleavage/ligation activities of the topoisomerase II. More generally, the steps pur-pur and pyr-pur are those presenting the highest BII contents. Relations are observed between the backbone phosphodiester BI/BII transitions and the flexibility of the deoxyribose sugar and the helical parameters such as roll. The roll is sequence dependent when the related phosphate is in the BI form, whereas this appears not to be true when it is in the BII form. The BI/BII transitions are associated with water migration, and new relations are observed with counterions. Indeed, it is observed that a strong coupling exists between the BII form and the presence of sodium ions near the adjacent sugar deoxyribose. The presence of sodium ions in the O4' surroundings or their binding could assist the BI to BII transition by furnishing energy. The implications of these new findings and, namely, their importance in the context of the sequence-dependent behavior of BI/BII transitions will be investigated in future studies.     

Combined vibrational,conformational energy and electron diffraction studies of trans-2-decalone

The structure of trans-2-decalone was investigated by combined gas phase electron diffraction, conformational energy and vibrational analyses. In this study first the minimum energy conformations for trans-2-decalone were calculated by molecular mechanics techniques using a force field described in the literature; the same field and the minimum energy conformations were then used in subsequent vibrational analyses to calculate the mean amplitudes of vibration for each minimum energy conformation of trans-2-decalone; these mean amplitudes and the corresponding internuclear distances were then used to calculate theoretical electron diffraction radial distribution curves which were compared to the experimental curves. Four conformational energy minima were investigated with one or both rings in a chair or non-chair form. The results of the combined investigations indicate that the molecule exists in the conformation which has both rings in a distorted chair form.     

Free energy landscape of A-DNA to B-DNA conversion in aqueous solution

The interconversion between the well-characterized A- and B-forms of DNA is a structural transition for which the intermediate states and the free energy difference between the two endpoints are not known precisely. In the present study, the difference between the Root Mean Square Distance (RMSD) from canonical A-form and B-form DNA is used as an order parameter to characterize this free energy difference using umbrella sampling molecular dynamics (MD) simulations with explicit solvent. The constraint imposed along this order parameter allows relatively unrestricted evolution of the intermediate structures away from both canonical A- and B-forms. The free energy difference between the A- and B-forms for the hexamer DNA sequence CTCGAG in aqueous solution is conservatively estimated to be at least 2.8 kcal/mol. A continuum of intermediate structures with no well-defined local minima links the two forms. The absence of any major barriers in the free energy surface is consistent with spontaneous conversion of the A-form DNA to B-form DNA in unconstrained simulations. The extensive sampling in the MD simulations (>0.1 mus) also allowed quantitative energetic characterization of local backbone conformational variables such as sugar pseudorotation angles and BI/BII state equilibria and their dependence on base identity. The absolute minimum in the calculated free energy profile corresponds closely to the crystal structure of the hexamer sequence, indicating that the present method has the potential to identify the most stable state for an arbitrary DNA sequence in water.     

Relaxed energetic maps of κ‐carrabiose: A DFT study

The B3LYP density function was used with the 6‐31G(d) basis set to perform relaxed energetic contour maps of the charged form of κ‐carrabiose in the gas phase and for the neutral form first in the gas phase and then by simulating the presence of water as solvent using the Onsager model. Only one starting conformation has been considered to perform all the calculations. Rigid energetic maps have been then constructed either by addition of diffuse or polarization functions to the basis set obtaining in that way 6‐31+G(d)//6‐31G(d), 6‐31+G(d,p)//6‐31G(d), and 6‐311++G(d,p)//6‐31G(d) energetic maps that have been carefully examined. The obtained structures corresponding to the lower energy conformers have been then fully optimized using different basis sets with the B3LYP method, a reversion in term of energy has been observed for the two first minima in the case of the charged disaccharide in the gas phase, this was attributed to the large grid of 30° that could lead to the exclusion of an intermediate value corresponding to the real minimum of energy. We thus suggest that after establishing potential energy maps it is essential to proceed to full optimizations of the lower energy conformers. Calculations using the more accurate correlated method MP2 with the 6‐31G(d) basis set have also been performed for conformers of the two disaccharides in the gas phase. © 2010 Wiley Periodicals, Inc. J Comput Chem 2010     

Insertion of a bulky rhodium complex into a DNA cytosine-cytosine mismatch: an NMR solution study

The bulky octahedral complex Rh(bpy)2chrysi3+ (chrysi = 5,6-chrysenequinonediimine) binds single-base mismatches in a DNA duplex with micromolar binding affinities and high selectivity. Here we present an NMR solution study to characterize the binding mode of this bulky metal complex with its target CC mismatch in the oligonucleotide duplex (5'-CGGACTCCG-3')2. Both NOESY and COSY studies indicate that Rh(bpy)2chrysi3+ inserts deeply in the DNA at the mismatch site via the minor groove and with ejection of both destabilized cytosines into the opposite major groove. The insertion only minimally distorts the conformation of the oligonucleotide local to the binding site. Both flanking, well-matched base pairs remain tightly hydrogen-bonded to each other, and 2D DQF-COSY experiments indicate that all sugars maintain their original C2'-endo conformation. Remarkably, 31P NMR reveals that opening of the phosphate angles from a BI to a BII conformation is sufficient for insertion of the bulky metal complex. These results corroborate those obtained crystallographically and, importantly, provide structural evidence for this specific insertion mode in solution.     

Quantum-Chemical and semiclassical calculations of intermolecular interactions of phospholipids

By use of empirical 0–1–6–12 atom–atom potential functions and the PCILOCC method intra- and intermolecular interactions of glycero–phosphoryl–ethanolamine model head groups in a planar layer crystal were calculated. Starting from investigations of the two-dimensional energy-contour diagrams the minima of energy as a function of all head group torsion angles were calculated using a gradient procedure. Within an interval of 15 kcal/mol above the energy of the global minimum we obtained about 30 local minima. These results demonstrate a high flexibility of the investigated phosphorylethanolamine head group in agreement with experiment. The ethanolamine moiety exists in enantiomeric conformations. With the torsion angles of the 0–1–6–12 energy minimization procedure PCILOCC calculations were carried out. These calculations yield the x-ray conformation as the most stable one (unit-cell stabilization energy = ?36.3 kcal/mol). The PCILOCC as well as the potential function calculations show that the conformation of phospholipid head groups in layer crystals is determined by intramolecular as well as by intermolecular interactions with neighboring phospholipid molecules.     

Flexible protein‐protein docking based on Best‐First search algorithm

We developed a new high resolution protein‐protein docking method based on Best‐First search algorithm that loosely imitates protein‐protein associations. The method operates in two stages: first, we perform a rigid search on the unbound proteins. Second, we search alternately on rigid and flexible degrees of freedom starting from multiple configurations from the rigid search. Both stages use heuristics added to the energy function, which causes the proteins to rapidly approach each other and remain adjacent, while optimizing on the energy. The method deals with backbone flexibility explicitly by searching over ensembles of conformations generated before docking. We ran the rigid docking stage on 66 complexes and grouped the results into four classes according to evaluation criteria used in Critical Assessment of Predicted Interactions (CAPRI; “high,” “medium,” “acceptable,” and “incorrect”). Our method found medium binding conformations for 26% of the complexes and acceptable for additional 44% among the top 10 configurations. Considering all the configurations, we found medium binding conformations for 55% of the complexes and acceptable for additional 39% of the complexes. Introducing side‐chains flexibility in the second stage improves the best found binding conformation but harms the ranking. However, introducing side‐chains and backbone flexibility improve both the best found binding conformation and the best found conformation in the top 10. Our approach is a basis for incorporating multiple flexible motions into protein‐protein docking and is of interest even with the current use of a simple energy function. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2010     

Racemic DNA Crystallography

Racemates increase the chances of crystallization by allowing molecular contacts to be formed in a greater number of ways. With the advent of protein synthesis, the production of protein racemates and racemic‐protein crystallography are now possible. Curiously, racemic DNA crystallography had not been investigated despite the commercial availability of L ‐ and D ‐deoxyribo‐oligonucleotides. Here, we report a study into racemic DNA crystallography showing the strong propensity of racemic DNA mixtures to form racemic crystals. We describe racemic crystal structures of various DNA sequences and folded conformations, including duplexes, quadruplexes, and a four‐way junction, showing that the advantages of racemic crystallography should extend to DNA.     

Racemic DNA Crystallography

Racemates increase the chances of crystallization by allowing molecular contacts to be formed in a greater number of ways. With the advent of protein synthesis, the production of protein racemates and racemic‐protein crystallography are now possible. Curiously, racemic DNA crystallography had not been investigated despite the commercial availability of L ‐ and D ‐deoxyribo‐oligonucleotides. Here, we report a study into racemic DNA crystallography showing the strong propensity of racemic DNA mixtures to form racemic crystals. We describe racemic crystal structures of various DNA sequences and folded conformations, including duplexes, quadruplexes, and a four‐way junction, showing that the advantages of racemic crystallography should extend to DNA.     

New insights in the electrocatalytic proton reduction and hydrogen oxidation by bioinspired catalysts: a DFT investigation

In this paper, we present a DFT study of the proton reduction mechanism catalyzed by the complex [Ni(P?(H)N?(H))?](2+), bioinspired from the hydrogenases. A detailed analysis of the reactive isomers is discussed together with the localizations of the transitions states and energy minima. The reactive catalytic species is a biprotonated Ni(0) complex that can show different conformations and that can be protonated on different sites. The energies of the different conformations and biprotonated species have been calculated and discussed. Energy barriers for two different reaction mechanisms have been identified in solvent and in gas phase. Frequency calculations have been performed to check the nature of the energy minima and for the calculations of entropic energetic terms and zero point energies. We show that only one conformation is mostly reactive. All the others species are nonreactive in their original form, and they have to pass through conformational barriers in order to transform in the reactive species.     

The N6-methyl group of adenine further increases the BI stability of DNA compared to C5-methyl groups

Methylated DNA bases are natural modifications which play an important role in protein-DNA interactions. Recent experimental and theoretical results have shown an influence of the base modification on the conformational behavior of the DNA backbone. MD simulations of four different B-DNA dodecamers (d(GC)(6), d(AT)(6), d(G(5mCG)(5)C), and d(A(T6mA)(5)T)) have been performed with the aim to examine the influence of methyl groups on the B-DNA backbone behavior. An additional control simulation of d(AU)(6) has also been performed to examine the further influence of the C5-methyl group in thymine. Methyl groups in the major groove (as in C5-methylcytosine, thymine, or N6-methyladenine) decrease the BII substate population of RpY steps. Due to methylation a clearer distinction of the BI substate stability between YpR and RpY (CpG/GpC or TpA/ApT) steps arises. A positive correlation between the BII substate population and base stacking distances is seen only for poly(GC). A methyl group added into the major groove increases mean water residence times around the purine N7 atom, which may stabilize the BI substate by improving the hydration network between the DNA backbone and the major groove. The N6-methyl group also forms a water molecule bridge between the N6 and O4 atoms, and thus further stabilizes the BI substate.     

Kinetics of cooperative protein folding involving two separate conformational families

The master equation that describes the kinetics of protein folding is solved numerically for a portion of Staphylococcal Protein A by a Laplace transformation. The calculations are carried out with 50 local-minimum conformations belonging to two conformational families. The master equation allows for transitions among all the 50 conformations in the evolution toward the final folded equilibrium distribution of conformations. It is concluded that the native protein folds in a fast cooperative process. The global energy minimum of a native protein can be reached after a sufficiently long folding time regardless of the initial state and the existence of a large number of local energy minima. Conformations representing non-native states of the protein can transform to the native state even if they do not belong to the native conformational family. Given a starting conformation, the protein molecule can fold to its final conformation through different paths. Finally, when the folding reaches the equilibrium distribution, the protein molecule adopts a set of conformations in which the global minimum has the largest average probability.     

HF/6‐31G* energy surfaces for disaccharide analogs

The HF/6‐31G* level of theory was used to calculate relaxed potential energy surfaces for 12 analogs of disaccharides. The analogs were made by replacing glucose with tetrahydropyran and fructose with 2‐methyltetrahydrofuran. Molecules had zero, one or two anomeric carbon atoms, and di‐axial, axial‐equatorial, and di‐equatorial linkages. Despite the absence of hydroxyl groups, the surfaces account well for conformations that are observed in crystals of the parent disaccharides. Thus, torsional energy and the simple bulk of ring structures are major factors in determining disaccharide conformation. The contour shapes around the global minima depend on the number of anomeric carbons involved in the linkage, while the presence of alternative minima that have relative energies less than 4 kcal/mol mostly requires equatorial bonds. However, molecules with two adjacent anomeric centers gave exceptions to these rules. Flexibility values related to a partition function show that the di‐axial trehalose analog is the most rigid. The di‐equatorial pseudodisaccharide analog with no anomeric centers is most flexible. Reproduction of these surfaces is proposed as a simple test of force fields for modeling carbohydrates. Also, these surfaces can be used in a simple hybrid method for calculating disaccharide energy surfaces. © 2000 John Wiley & Sons, Inc. * J Comput Chem 22: 65–78, 2001     

31P NMR investigation of backbone dynamics in DNA binding sites

The backbone conformation of DNA plays an important role in the indirect readout mechanisms for protein--DNA recognition events. Thus, investigating the backbone dynamics of each step in DNA binding sequences provides useful information necessary for the characterization of these interactions. Here, we use 31P dynamic NMR to characterize the backbone conformation and dynamics in the Dickerson dodecamer, a sequence containing the EcoRI binding site, and confirm solid-state 2H NMR results showing that the C3pG4 and C9pG10 steps experience unique dynamics and that these dynamics are quenched upon cytosine methylation. In addition, we show that cytosine methylation affects the conformation and dynamics of neighboring nucleotide steps, but this effect is localized to only near neighbors and base-pairing partners. Last, we have been able to characterize the percent BII in each backbone step and illustrate that the C3pG4 and C9pG10 favor the noncanonical BII conformation, even at low temperatures. Our results demonstrate that 31P dynamic NMR provides a robust and efficient method for characterizing the backbone dynamics in DNA. This allows simple, rapid determination of sequence-dependent dynamical information, providing a useful method for studying trends in protein-DNA recognition events.     

Exploring the similarities between potential smoothing and simulated annealing

Simulated annealing and potential function smoothing are two widely used approaches for global energy optimization of molecular systems. Potential smoothing as implemented in the diffusion equation method has been applied to study partitioning of the potential energy surface (PES) for N‐Acetyl‐Ala‐Ala‐N‐Methylamide (CDAP) and the clustering of conformations on deformed surfaces. A deformable version of the united‐atom OPLS force field is described, and used to locate all local minima and conformational transition states on the CDAP surface. It is shown that the smoothing process clusters conformations in a manner consistent with the inherent structure of the undeformed PES. Smoothing deforms the original surface in three ways: structural shifting of individual minima, merging of adjacent minima, and energy crossings between unrelated minima. A master equation approach and explicit molecular dynamics trajectories are used to uncover similar features in the equilibrium probability distribution of CDAP minima as a function of temperature. Qualitative and quantitative correlations between the simulated annealing and potential smoothing approaches to enhanced conformational sampling are established. © 2000 John Wiley & Sons, Inc. J Comput Chem 21: 531–552, 2000     

Hydrogen bonding and the conformations of poly(alkyl acrylamides)

The conformations of poly(alkyl acrylamide) oligomers in nonpolar solvents were studied using molecular dynamics techniques. Poly(methyl acrylamide) was found to collapse to a globule-like conformation at low temperatures; however, excluded volume effects inhibited the collapse of poly(octadecyl acrylamide). A high density of structured units, characterized by a trans-gauche-trans-trans-gauche-trans torsional sequence along the backbone, was noted in all simulations. Such units were found to create a particularly stable set of intramolecular hydrogen bonds. An oligomer constructed with these stable units was found to have significantly lower minimized energy than both the all-trans and the helical backbone conformations. The constructed conformation had lower Coulomb energy (more hydrogen bonds) than the all-trans conformation and lower dihedral energy (less backbone distortion) than the helical conformation. The propensity for poly(octadecyl acrylamide) to form hydrogen bonds introduced significant disorder into the orientation of the alkyl side chains. This disorder would inhibit crystallization and restrict the ability of such polymers to form epitaxial seeds for nucleating paraffin crystals.     

Multiple binding modes of the camptothecin family to DNA oligomers

The binding constants of camptothecin, topotecan and its lactone ring-opened carboxylate derivative to DNA octamers were measured by UV and NMR spectroscopy. The self-association of topotecan (TPT) was also measured. The carboxylate form of TPT binds in the same way as the lactone, but more weakly. Titration of TPT into d(GCGATCGC)2 shows a preferred location stacked onto the terminal G1 base. However, the intermolecular NOEs cannot be reconciled with a single conformation of the complex, and suggest a model of a limited number of conformations in fast exchange. MD calculations on four pairs of starting structures with TPT stacked onto the G1-C8 base pair in different orientations were therefore performed. The use of selected experimental "docking" restraints yielded ten MD trajectories covering a wide conformational space. From a combination of calculated free energies, NOEs and chemical shifts, some of the structures produced could be eliminated, and it is concluded that the data are consistent with two major families of conformations in fast exchange. One of these is the conformation found in a crystal of a TPT/DNA/topoisomerase I ternary complex [Proc. Natl. Acad. Sci. USA 2002, 99, 15 387-15 392].     

Quantification of DNA BI/BII backbone states in solution. Implications for DNA overall structure and recognition

The backbone states of B-DNA influence its helical parameters, groove dimensions, and overall curvature. Therefore, detection and fine characterization of these conformational states are desirable. Using routine NMR experiments on a nonlabeled B-DNA oligomer and analyzing high-resolution X-ray structures, we investigated the relationship between interproton distances and backbone conformational states. The three H2'i-H6/8i+1, H2' 'i-H6/8i+1, and H6/8i-H6/8i+1 sequential distances were found cross-correlated and linearly coupled to epsilon-zeta values in X-ray structures and 31P chemical shifts (deltaP) in NMR that reflect the interconversion between the backbone BI (epsilon-zeta < 0 degrees ) and BII (epsilon-zeta > 0 degrees) states. These relationships provide a detailed check of the NMR data consistency and the possibility to extend the set of restraints for structural refinement through various extrapolations. Furthermore, they allow translation of deltaP in terms of BI/BII ratios. Also, comparison of many published deltaP in solution to crystal data shows that the impact of sequence on the BI/BII propensities is similar in both environments and is therefore an intrinsic and general property of B-DNA. This quantification of the populations of BI and BII is of general interest because these sequence-dependent backbone states act on DNA overall structure, a key feature for DNA-protein-specific recognition.