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.     

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.     

DFT study of polymorphism of the DNA double helix at the level of dinucleoside monophosphates

We apply DFT calculations to deoxydinucleoside monophosphates (dDMPs) which represent minimal fragments of the DNA chain to study the molecular basis of stability of the DNA duplex, the origin of its polymorphism and conformational heterogeneity. In this work, we continue our previous studies of dDMPs where we detected internal energy minima corresponding to the “classical” B conformation (BI‐form), which is the dominant form in the crystals of oligonucleotide duplexes. We obtained BI local energy minima for all existing base sequences of dDMPs. In the present study, we extend our analysis to other families of DNA conformations, successfully identifying A, BI, and BII energy minima for all dDMP sequences. These conformations demonstrate distinct differences in sugar ring puckering, but similar sequence‐dependent base arrangements. Internal energies of BI and BII conformers are close to each other for nearly all the base sequences. The dGpdG, dTpdG, and dCpdA dDMPs slightly favor the BII conformation, which agrees with these sequences being more frequently experimentally encountered in the BII form. We have found BII‐like structures of dDMPs for the base sequences both existing in crystals in BII conformation and those not yet encountered in crystals till now. On the other hand, we failed to obtain dDMP energy minima corresponding to the Z family of DNA conformations, thus giving us the ground to conclude that these conformations are stabilized in both crystals and solutions by external factors, presumably by interactions with various components of the media. Overall the accumulated computational data demonstrate that the A, BI, and BII families of DNA conformations originate from the corresponding local energy minimum conformations of dDMPs, thus determining structural stability of a single DNA strand during the processes of unwinding and rewinding of DNA. © 2010 Wiley Periodicals, Inc. Int J Quantum Chem 110:2548–2559, 2010     

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.     

Interpreting protein structural dynamics from NMR chemical shifts

In this investigation, semiempirical NMR chemical shift prediction methods are used to evaluate the dynamically averaged values of backbone chemical shifts obtained from unbiased molecular dynamics (MD) simulations of proteins. MD-averaged chemical shift predictions generally improve agreement with experimental values when compared to predictions made from static X-ray structures. Improved chemical shift predictions result from population-weighted sampling of multiple conformational states and from sampling smaller fluctuations within conformational basins. Improved chemical shift predictions also result from discrete changes to conformations observed in X-ray structures, which may result from crystal contacts, and are not always reflective of conformational dynamics in solution. Chemical shifts are sensitive reporters of fluctuations in backbone and side chain torsional angles, and averaged (1)H chemical shifts are particularly sensitive reporters of fluctuations in aromatic ring positions and geometries of hydrogen bonds. In addition, poor predictions of MD-averaged chemical shifts can identify spurious conformations and motions observed in MD simulations that may result from force field deficiencies or insufficient sampling and can also suggest subsets of conformational space that are more consistent with experimental data. These results suggest that the analysis of dynamically averaged NMR chemical shifts from MD simulations can serve as a powerful approach for characterizing protein motions in atomistic detail.     

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.     

Enforcing periodic secondary structures in hybrid peptides: a novel hybrid foldamer containing periodic gamma-turn motifs

This note describes the design, synthesis, and conformational studies of a novel hybrid foldamer that adopts a definite compact, three-dimensional structure determined by a combined effect of the special conformational properties of the foldamer constituents. The striking feature of this de novo designed foldamer is its ability to display periodic gamma-turn conformations stabilized by intramolecular hydrogen bonds. Conformational investigations by single-crystal X-ray studies, solution-state NMR, and ab initio MO theory at the HF/6-31G* level strongly support the prevalence of gamma-turn motifs in both the di- and tetrapeptide foldamers, which are presumably stabilized by bifurcated hydrogen bonds in the solid and solution states. The strategy disclosed herein for the construction of hybrid foldamers with periodic gamma-turn motifs has the potential to significantly augment the conformational space available for foldamer design with diverse backbone structures and conformations.     

Beta-Ala containing peptides: potentials in design and construction of bioactive peptides and protein secondary structure mimics

In recent years, there has been increasing interest in de novo design and construction of novel synthetic peptides that mimic protein secondary structures, i.e., turns, helices and sheets. The unique structural influences exerted by unsubstituted, non-coded, non-chiral beta-amino acid, i.e., beta-alanine (beta-Ala; 3- or beta- aminopropionic acid) on peptide backbone, when inserted into peptide chain comprised alpha-amino acids, offer an excellent opportunity to design and construct diverse well-defined three-dimensional structures. Our current understanding of folding-unfolding behavior of the beta-Ala residues relies primarily from an examination of conformational preferences of a large number of short cyclic- as well as acyclic beta-Ala containing peptides investigated using single crystal X-ray diffraction analysis. In addition, theoretical conformational energy calculations and different spectroscopic techniques: 1H NMR, FT-IR and CD, have also been employed although, to a lesser extent. The obtainable results tend to reveal overwhelming preferences of the beta-Ala moiety for the folded gauche (mu approximately +/-65+/-10 degrees conformation in cyclic- and for an extended trans (mu approximately +/-165+/-10 degrees) as well as gauche (mu approximately +/-65+/-10 degrees) orientations in acyclic beta-Ala containing peptides. The results also indicate that in short linear beta-Ala containing peptides, the specific influence of selective neighboring side-chain substituents e.g. linear- or cyclic symmetrically C(alpha,alpha)-disubstituted glycines and other conformational constraints, may be significant in controlling the overall folded-unfolded topographical features across the two methylene units (-CbetaH2-CalphaH2-) of the beta-Ala residue. Taking into consideration the wide occurrence of beta-Ala moiety in animal and plant kingdoms and the remarkable structural versatility of the peptides incorporating beta-Ala residue(s), together with appreciable resistance towards enzymatic degradation, hold strong promise for biophysicists and biochemists not only to design molecules that fold to mimic protein secondary structures but also to develop potent peptide analogs and peptidomimetics displaying unique pharmaceutical properties.     

Synthesis and conformational study of 1,3,2-oxazaphosphorino[4, 3-a]isoquinolines: a new ring system

A set of 1,3,2-oxazaphosphorino[4,3-a]isoquinolines 6a,b-9a,b, a new ring system, was synthesized, and their stereochemical and conformational analyses were performed by (1)H, (13)C, and (31)P NMR methods. X-ray measurements were also carried out to confirm the stereochemical assignments and conformational results obtained by means of NMR. Intermediate coupling constants (3)J(P,H) were found for compounds 7 and 9; these do not relate to equilibria between previously reported conformers, but are indicative of new distorted conformational states in solution. The connecting isoquinoline and the steric interaction between the aromatic moiety and the Me-1 substituent can block the oxazaphosphorinane ring. The conformational behavior of compounds 6 and 8 was characterized by the usual chair-twist equilibrium.     

A ribose sugar conformational switch in the LTR-retrotransposon Ty3 polypurine tract-containing RNA/DNA hybrid

To probe structural features of a polypurine tract (PPT) that mediate its specific recognition and processing, a model 20 bp RNA/DNA hybrid duplex, which includes the full PPT sequence of the Saccharomyces cerevisiae LTR-retrotransposon Ty3, has been investigated using solution NMR spectroscopy. While homonuclear NOESY and DQF-COSY analyses indicate that this PPT-containing RNA/DNA hybrid adopts an overall A-form-like helical geometry, an unexpected sugar pucker switch has been detected for the ribose at position +1, relative to the cleavage site, on the RNA strand. A model of the conformational changes induced by the A- to B-type sugar pucker switch shows a significant change in the backbone trajectory of the RNA strand, which alters the presentation of backbone phosphate and 2' hydroxyl groups 3' of this residue. This observation implies that part of the mechanism governing RNase H fidelity may be through distortion of the RNA/DNA helix one base ahead of the scissile bond.     

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.     

The synthesis and reactivity of a molybdenum (IV) stretched-dihydrogen complex

Reaction of [Mo(NPh)(PMe3)3(o-(Me3SiN)2C6H4)] (1) with molecular hydrogen (ca. 1 atm) at -10 degrees C in toluene-d8 results in the formation of dihydrogen complex [Mo(NPh)(PMe3)2(H2)(o-(Me3SiN)2C6H4)] (2) by 1H and 31P NMR spectroscopy. In solution at -50 degrees C 1 and 2 are present in a 1:3 ratio, respectively. The nature of dihydrogen ligand bonding in 2 was probed by T1 analysis and analysis of the JH-D coupling constant in the deuterium hydride isotopomer of 2 giving H-H distances of 1.18 A and 1.17 A, respectively. When allowed to warm to 30 degrees C, 2 reacts affording [Mo(NPh)(PMe3)3(o-(Me3SiN)(NH)C6H4)] (3) over a 1 h period. The X-ray structures of 1 and 3 are reported.     

The molecular electrostatic potential of the B-DNA helix

The overlap multipole expansion procedure is utilized for the evaluation of the component of the electrostatic molecular potential of the B-DNA helix due to its sugar-phosphate backbone. The overall shape of the potential, its extension in space, the location of the minima and the differences in the values of the potential in particularly significant regions (minor and major grooves, vicinity of the phosphates) are indicated. The isopotential surfaces are practically cylindrical at distances larger than 15 Å from the helix axis but exhibit a more complex structure at shorter distances.This paper is dedicated to Professor H. Hartmann on the occasion of his 65th birthday.     

C5-methylation of cytosine in B-DNA thermodynamically and kinetically stabilizes BI

We have examined the backbone dynamics of two alternating purine-pyrimidine dodecamers. One sequence consists of "pure" GC bases; the other one contains 5-methylcytosines. The effect of the methyl groups on the backbone substates BI/BII was investigated by means of molecular dynamics. The methylation influences, on one hand, the transition barrier between BI and BII and, on the other hand, the state of equilibrium. The kinetic consequences are an increase of the DeltaG of Gp5mC steps by 1.5 kcal/mol and a decrease of the DeltaG of 5mCpG steps by 0.8 kcal/mol (compared with the nonmethylated DNA). Thus, the additive group differentiates between the two occurring dinucleotide steps and renders the phosphate of the 5-methylcytosine more rigid, as proposed by experimental studies. The thermodynamic consequences are an increase of the DeltaG of Gp5mC steps by 1.1 kcal/mol and a decrease of the DeltaG of 5mCpG steps by 0.8 kcal/mol. The reason for this shift in equilibrium is still not completely clear on a molecular basis. But we can conclude that the indirect readout of DNA is influenced by methylation.     

Pyridin-2-yl guanidine derivatives: conformational control induced by intramolecular hydrogen-bonding interactions

The synthesis and conformational analysis of a series of pyridin-2-yl guanidine derivatives using NMR, X-ray crystallography, and B3LYP/6-31+G** theoretical studies are reported. A remarkable difference was observed in the (1)H NMR spectra of the guanidinium salts as compared with their N,N'-di-Boc protected and neutral analogues. This difference corresponds to a 180° change in the dihedral angle between the guanidine/ium moiety and the pyridine ring in the salts as compared to the Boc-protected derivatives, a conclusion that was supported by theoretical studies, X-ray data, and NMR analysis. Moreover, our data sustain the existence of two intramolecular hydrogen-bonding systems: (i) between the pyridine N1 atom and the guanidinium protons in the salts and (ii) within the tert-butyl carbamate groups of the Boc-protected derivatives. To verify that the observed conformational control arises from these intramolecular interactions, a new series of N-Boc-N'-propyl-substituted pyridin-2-yl guanidines were also prepared and studied.     

High-temperature, high-pressure hydrothermal synthesis, crystal structure, and solid-state NMR spectroscopy of Cs2(UO2)(Si2O6) and variable-temperature powder X-ray diffraction study of the hydrate phase Cs2(UO2)(Si2O6) x 0.5H2O

A new uranium(VI) silicate, Cs2(UO2)(Si2O6), has been synthesized by a high-temperature, high-pressure hydrothermal method and characterized by single-crystal X-ray diffraction and solid-state NMR spectroscopy. It crystallizes in the orthorhombic space group Ibca (No. 73) with a = 15.137(1) A, b = 15.295(1) A, c = 16.401(1) A, and Z = 16. Its structure consists of corrugated achter single chains of silicate tetrahedra extending along the c axis linked together via corner-sharing by UO6 tetragonal bipyramids to form a 3-D framework which delimits 8- and 6-ring channels. The Cs+ cations are located in the channels or at sites between channels. The 29Si and 133Cs MAS NMR spectra are consistent with the crystal structure as determined from X-ray diffraction, and the resonances in the spectra are assigned. Variable-temperature in situ powder X-ray diffraction study of the hydrate Cs2(UO2)(Si2O6) x 0.5H2O indicates that the framework structure is stable up to 800 degrees C and transforms to the structure of the title compound at 900 degrees C. A comparison of related uranyl silicate structures is made.     

Nonadiabatic effects on peptide vibrational dynamics induced by conformational changes

Quantum dynamical simulations of vibrational spectroscopy have been carried out for glycine dipeptide (CH(3)-CO-NH-CH(2)-CO-NH-CH(3)). Conformational structure and dynamics are modeled in terms of the two Ramachandran dihedral angles of the molecular backbone. Potential energy surfaces and harmonic frequencies are obtained from electronic structure calculations at the density functional theory (DFT) [B3LYP/6-31+G(d)] level. The ordering of the energetically most stable isomers (C(7) and C(5)) is reversed upon inclusion of the quantum mechanical zero point vibrational energy. Vibrational spectra of various isomers show distinct differences, mainly in the region of the amide modes, thereby relating conformational structures and vibrational spectra. Conformational dynamics is modeled by propagation of quantum mechanical wave packets. Assuming a directed energy transfer to the torsional degrees of freedom, transitions between the C(7) and C(5) minimum energy structures occur on a sub-picosecond time scale (700...800 fs). Vibrationally nonadiabatic effects are investigated for the case of the coupled, fundamentally excited amide I states. Using a two state-two mode model, the resulting wave packet dynamics is found to be strongly nonadiabatic due to the presence of a seam of the two potential energy surfaces. Initially prepared adiabatic vibrational states decay upon conformational change on a time scale of 200...500 fs with population transfer of more than 50% between the coupled amide I states. Also the vibrational energy transport between localized (excitonic) amide I vibrational states is strongly influenced by torsional dynamics of the molecular backbone where both enhanced and reduced decay rates are found. All these observations should allow the detection of conformational changes by means of time-dependent vibrational spectroscopy.     

Low-energy electron diffraction and induced damage in hydrated DNA

Elastic scattering of 5-30 eV electrons within the B-DNA 5'-CCGGCGCCGG-3' and A-DNA 5'-CGCGAATTCGCG-3' DNA sequences is calculated using the separable representation of a free-space electron propagator and a curved wave multiple scattering formalism. The disorder brought about by the surrounding water and helical base stacking leads to a featureless amplitude buildup of elastically scattered electrons on the sugar and phosphate groups for all energies between 5 and 30 eV. However, some constructive interference features arising from diffraction are revealed when examining the structural waters within the major groove. These appear at 5-10, 12-18, and 22-28 eV for the B-DNA target and at 7-11, 12-18, and 18-25 eV for the A-DNA target. Although the diffraction depends on the base-pair sequence, the energy dependent elastic scattering features are primarily associated with the structural water molecules localized within 8-10 A spheres surrounding the bases and/or the sugar-phosphate backbone. The electron density buildup occurs in energy regimes associated with dissociative electron attachment resonances, direct electronic excitation, and dissociative ionization. Since diffraction intensity can be localized on structural water, compound H2O:DNA states may contribute to energy dependent low-energy electron induced single and double strand breaks.     

Sequence-structure relationships in DNA oligomers: a computational approach

A collective-variable model for DNA structure is used to predict the conformation of a set of 30 octamer, decamer, and dodecamer oligomers for which high-resolution crystal structures are available. The model combines an all-atom base pair representation with an empirical backbone, emphasizing the role of base stacking in fixing sequence-dependent structure. We are able to reproduce trends in roll and twist to within 5 degrees across a large database of both A- and B-DNA oligomers. A genetic algorithm approach is used to search for global minimum structures and this is augmented by a grid search to identify local minimums. We find that the number of local minimums is highly sequence dependent, with certain sequences having a set of minimums that span the entire range between canonical A- and B-DNA conformations. Although the global minimum does not always agree with the crystal structure, for 24 of the 30 oligomers, we find low-energy local minimums that match the experimental step parameters. Discrepancies throw some light on the role of crystal packing in determining the solid-state conformation of double-helical DNA.     

A conformational study of the trisaccharide beta-D-Glcp-(1-->2)[beta-D-Glcp-(1-->3)]alpha-D-Glcp-OMe by NMR NOESY and TROESY experiments, computer simulations, and X-ray crystal structure analysis

Proton-proton cross-relaxation rates have been measured for the trisaccharide beta-D-Glcp-(l --> 2)[beta-D-Glcp-(1 --> 3)]alpha-D-Glcp-OMe in D2O as well as in D2O/[D6]DMSO 7:3 solution at 30 degrees C by means of one-dimensional NMR pulsed field gradient 1H,1H NOESY and TROESY experiments. Interatomic distances for the trisaccharide in D2O were calculated from the cross-relaxation rates for two intraresidue and three interglycosidic proton pairs, using the isolated spin-pair approximation. In the solvent mixture one intraresidue and three interglycosidic distances were derived without the use of a specific molecular model. In this case the distances were calculated from the cross-relaxation rates in combination with "model-free" motional parameters previously derived from 13C relaxation measurements. The proton-proton distances for interglycosidic pairs were compared with those averaged from Metropolis Monte Carlo and Langevin Dynamics simulations with the HSEA, PARM22, and CHEAT95 force fields. The crystal structure of the trisaccharide was solved by analysis of X-ray data. Interresidue proton pairs from the crystal structure and those observed by NMR experiments were similar. However, the corresponding proton-proton distances generated by computer simulations were longer. For the (1 --> 2) linkage the glycosidic torsion angles of the crystal structure were found in a region of conformational space populated by all three force fields, whereas for the (1 --> 3) linkage they occupied a region of low population density, as seen from the simulations.