Sequence-dependent anisotropic flexibility of B-DNA. A conformational study

Bending flexibility of the six tetrameric duplexes was investigated d(AAAA):d(TTTT), d(AATT)2, d(TTAA)2, d(GGGG):d(CCCC), d(GGCC)2 and d(CCGG)2,. The tetramers were extended in the both directions by regular double helices. The stiffness of the B-DNA double helix when bent into the both grooves proved to be less than that in the perpendicular direction by an order of magnitude. Such an anisotropy is a property of the sugar-phosphate backbone structure. The calculated fluctuations of the DNA bending along the dyad axis, 5-7 degree, are in agreement with experimental value of the DNA persistence length. Anisotropy of the double helix is sequence-dependent: most easily bent into the minor groove are the tetramers with purine-pyrimidine dimer (RY) in the middle. In contrast, YR dinucleotides prefer bending into the major groove. Moreover, they have an equilibrium bend of 6-12 degree into this groove. The above inequality is caused by stacking interaction of the bases. The bend in the central dimer is distributed to some extent between the adjacent links, though the main fraction of the bend remains within the central link. Variation of the sugar-phosphate geometry in the bent helix is inessential, so that DNA remains within the B-family of forms: namely, when the helical axis is bent by 20 degree. the backbone dihedral angles vary by no more than 15 degree. The obtained results are in accord with x-ray structure of the B-DNA dodecamer; they further substantiate our early model of DNA wrapping in the nucleosome by means of mini-kinks separated by a half-pitch of the double helix, i.e. by 5-6 b.p. Sequence-dependent anisotropy of DNA presumably dictates the three-dimensional structure of DNA in solution as well. We have found that nonrandom allocation of YR dimers leads to the systematic bends in equilibrium structure of certain DNA fragments.     

Recent advances in the study of nucleic acid flexibility by molecular dynamics

The recent use of molecular dynamics (MD) simulations to study flexibility of nucleic acids has been reviewed from an analysis of the publications appearing in the past two years (from 2005 till date). Despite the existence of some unsolved problems in the methodologies, these years have been witness to major advances in the field. Based on a critical review of the most recent contributions, excitement exists on the expected evolution of the field in the next years.     

Towards a molecular dynamics consensus view of B-DNA flexibility

We present a systematic study of B-DNA flexibility in aqueous solution using long-scale molecular dynamics simulations with the two more recent versions of nucleic acids force fields (CHARMM27 and parmbsc0) using four long duplexes designed to contain several copies of each individual base pair step. Our study highlights some differences between pambsc0 and CHARMM27 families of simulations, but also extensive agreement in the representation of DNA flexibility. We also performed additional simulations with the older AMBER force fields parm94 and parm99, corrected for non-canonical backbone flips. Taken together, the results allow us to draw for the first time a consensus molecular dynamics picture of B-DNA flexibility.     

A theoretical study of the sequence specificity in binding of lexitropsins to B-DNA

A theoretical study is presented on the binding to B-DNA of a series of lexitropsins, these ligands being netropsin derivatives in which one or both of the pyrrole rings have been replaced by imidazoles. The best complexes have been located by energy minimisation taking into account nucleic acid flexibility, ligand flexibility, explicit, mobile counterions and solvent dielectric effects. Calculations have been performed for two homopolymeric DNA receptor sequences, AT base sequence, which only decreases in the imidazole derivatives. These results emphasize the decisive role of the molecular electrostatic potential of the nucleic acid in determining the sequence selectivity of these ligands, as opposed to the postulated role of adenine C2 - pyrrole beta hydrogen contacts.     

Molecular-modeling calculations of enzymatic enantioselectivity taking hydration into account

A new molecular-modeling methodology has been applied to explain enzymatic enantioselectivity in water. This methodology, which combines vacuum molecular mechanics and the continuum solvation method, should provide a more realistic view of the solvent-enzyme and solvent-substrate interactions than the heretofore used approaches involving the vacuum molecular mechanics only. The methodology described herein has been validated using the experimental data on alpha-chymotrypsin's enantioselectivity in the hydrolysis of four chiral substrates. The reasons why the vacuum molecular mechanics, although not taking hydration into account, still in most cases provide a satisfactory approximation of reality are discussed.     

Spermine-nucleic acid interactions: a theoretical study

The interaction of spermine with nucleic acids is simulated theoretically using refined semi-empirical energy formulae and an advanced minimization procedure. Various nucleic acids are considered: model homopolymeric DNA's, a dodecamer (CGCGAATTCGCG) of type B-DNA, as well as a transfer RNA, tRNA^Phe. The dominant role of electrostatic potential in determining the preferential binding sites of spermine is demonstrated in each of these cases and the role of counterions, nucleic acid structure, and base-pair sequence is analyzed.     

Observation of an A-DNA to B-DNA transition in a nonhelical nucleic acid hairpin molecule using molecular dynamics.

One of the truly challenging problems for molecular dynamics (MD) simulations is demonstrating that the trajectories can sample not only in the vicinity of an experimentally determined structure, but also that the trajectories can find the correct experimental structure starting from some other structure. Frequently these transitions to the correct structure require that the simulations overcome energetic barriers to conformational change. Here we present unrestrained molecular dynamics simulations of the DNA analogs of the RNA 5'-GGACUUCGGUCC-3' hairpin tetraloop. In one simulation we have used deoxyuracil residues, and in the other we have used the native DNA deoxythymine residues. We demonstrate that, on a nanosecond time scale, MD is able to simulate the transitions of both of the A-DNA stems to B-DNA stems within the constraints imposed by the four-base loop that caps the helix. These results suggest that we are now in a position to use MD to address the nature of sequence-dependent structural effects in nonduplex DNA structures.     

Quinoxaline antibiotics enhance peptide nucleic acid binding to double-stranded DNA

The effects of a wide range of DNA binding drugs on peptide nucleic acid (PNA) binding to double-stranded DNA by strand displacement have been investigated using a gel retardation assay. The bis-PNA [H-(Lys)-TTJTTJTTTT-(eg)(3)-TTTTCTTCTT-Lys-NH(2)] was used together with a 248 bp DNA fragment containing an appropriate target for the PNA. Most of the ligands that were studied, including DNA minor groove binders as well as intercalators and bis-intercalators, either have no effect or strongly inhibit PNA binding to DNA. By contrast, quinoxaline antibiotics facilitate PNA-DNA complex formation. The "PNA-helper" effect of echinomycin was studied in more detail using time and temperature dependence experiments to elucidate the mechanism. PNA binding to DNA follows pseudo-first-order kinetics, but the initial rate of binding is accelerated more than 10-fold in the presence of 10 microM echinomycin. The activation energy for PNA binding to dsDNA is lowered 2-fold by the antibiotic (45 vs 90 kJ/mol in the control). The reasons why quinoxalines promote the binding of PNA to DNA are not entirely clear but may well include distortions (opening) of the double helix that facilitate PNA invasion. This study establishes that the efficacy of DNA-targeted PNA antigene molecules could potentially be enhanced by judiciously adding certain DNA-interactive ligands.     

The flexibility of the nucleic acids: (III). The interaction of an aliphatic diamine, putrescine, with flexible B-DNA

A theoretical modelling of the interaction of putrescine (H3+N-(CH2)4-(+NH3) with DNA is carried out, introducing two new features which make the simulation of this interaction considerably more realistic. Firstly, the DNA to which putrescine is bound is fully flexible and thus able to respond to the distorting influence of the ligand. Secondly, the effect of changing the ratio of DNA base pairs per bound ligand is explicitly modelled. In this way, we have been able to confirm the experimentally known preference of putrescine binding with AT base pairs in B-DNA, but we also show, through the new features introduced, that the nature of the binding site of the ligand and the resulting impact on DNA conformation is strongly modified by the ligand binding density.     

Modelling of continuous microbial cultivation taking into account the memory effects

The problem of chemostat dynamics modelling for the purpose of control is considered. The "memory" of the culture is explicitly taken into account. Two possibilities for improving the quality of the proposed modelling approaches are discussed. A general model that accounts for the culture `memory' by means of different `memory' functions in the expressions of the specific growth rate and of the specific consumption rate and a polynomial function of the substrate concentration for the yield factor is proposed. The case where the maintenance energy is taken into account is also discussed. Two modifications of the general model (w-type and S-type) are presented. A zero-order `memory' function and a i-function with delay are applied in order to describe the `memory' effects. Continuous growth of the strain Saccharomyces cerevisiae on a glucose limited medium is considered as a case study. Detailed investigations of the variety of models, derived from the general model by applying different `memory' functions and different assumptions are carried out. The results are compared with those previously reported for the same process. It is shown that a significant improvement in predicting the substrate dynamics (not accompanied by any decrease in the quality of the model with respect to the biomass concentration) could be achieved, involving a first- or second-order polynomial function for the yield factor. It is also shown that the quality of the model mainly depends on the way that `memory' function is incorporated. The detailed investigations give priority to the w-type models. In this case past values of both biomass and substrate variables are considered. The time delay models with pure (constant) delay and those which account for the culture `memory' by zero-order `memory' function (adaptability parameter) are compared with respect to their utilization for the purpose of model-based control.     

Interactions of molecules with nucleic acids. I. An algorithm to generate nucleic acid structures with an application to the B-DNA structure and a counterclockwise helix.

An algorithm is developed that enables the routine determination of backbone conformations of nucleic acids. All atomic positions including hydrogen are specified in accord with experimental bond lengths and angles but with theoretically determined conformational angles. For two Watson-Crick base pairs at a separation of 3.38 Å, and perpendicular to a common helical axis, minimum energy configurations are found for all 10 combinations at helical angles of α ～ 36°–38°, corresponding to the B-DNA structure with C(2′)-endo sugar puckers. Backbone configurations exist only within the range 35.5° ? α ? 42°, which suggests the origin of the 10-fold helix. Calculated stacking energies for the B-DNA structure increases for each of the clustered groups of base pairs: G·C with G·C, G·C with A·T, and A·T with A·T, and they are in approximate agreement with experimental observations. The counter-clockwise helix is examined, and physically meaningful structures are found only when the helical axes of successive base pairs are disjointed.     

Mechanism of mycolic acid cyclopropane synthase: a theoretical study

The reaction mechanism of mycolic acid cyclopropane synthase is investigated using hybrid density functional theory. The direct methylation mechanism is examined with a large model of the active site constructed on the basis of the crystal structure of the native enzyme. The important active site residue Glu140 is modeled in both ionized and neutral forms. We demonstrate that the reaction starts via the transfer of a methyl to the substrate double bond, followed by the transfer of a proton from the methyl cation to the bicarbonate present in the active site. The first step is calculated to be rate-limiting, in agreement with experimental kinetic results. The protonation state of Glu140 has a rather weak influence on the reaction energetics. In addition to the natural reaction, a possible side reaction, namely a carbocation rearrangement, is also considered and is shown to have a low barrier. Finally, the energetics for the sulfur ylide proposal, which has already been ruled out, is also estimated, showing a large energetic penalty for ylide formation.     

Management of athletic training taking into account individual heart rate variability characteristics

This article presents a new approach to planning and timely adjusting athletic trainings according to the data of the quick analysis of heart rate variability. It has been shown that individual types of regulation are different not only in the autonomic balance, but also in the degree of endurance of training and competition loads.     

Salicylic acid is not a bacterial siderophore: a theoretical study

Using a newly available program for calculating the concentrations and speciation of various ions (Pettit, LD & Powell KJ, `SolEq' Academic Software, 1999), we have calculated that at pH 7 the amount of free Fe(III) present in an aqueous solution is 1.4×10^–9 M and not 10^–18 M as is usually quoted. In the presence of salicylic acid, included in the calculations at 10^–4 M, the solubility of Fe(III) is increased to only 9.8×10^–9 M suggesting that salicylate is unable to act as a siderophore although it is produced as an extracellular product by several bacterial genera when grown iron deficiently. In the presence of 40 mM phosphate, the soluble Fe(III) concentration is decreased by 10⁴ at pH 7 and again this is hardly affected by the presence of salicylate. Thus, for microorganisms grown either in vitro or in vivo, salicylate is unlikely to function as a iron solubilizing agent. The same conclusions may also apply to 2,3-dihydroxybenzoic acid.     

An extension to the metabolic control theory taking into account correlations between enzyme concentrations.

The classical metabolic control theory [Kacser, H. & Burns, J.A. (1973) Symp. Soc. Exp. Biol.27, 65-104; Heinrich, R. & Rapoport, T. (1974) Eur. J. Biochem.42, 89-95.] does not take into account experimental evidence for correlations between enzyme concentrations in the cell. We investigated the implications of two causes of linear correlations: competition between enzymes, which is a mere physical adaptation of the cell to the limitation of resources and space, and regulatory correlations, which result from the existence of regulatory networks. These correlations generate redistribution of enzyme concentrations when the concentration of an enzyme varies; this may dramatically alter the flux and metabolite concentration curves. In particular, negative correlations cause the flux to have a maximum value for a defined distribution of enzyme concentrations. Redistribution coefficients of enzyme concentrations allowed us to calculate the 'combined response coefficient' that quantifies the response of flux or metabolite concentration to a perturbation of enzyme concentration.     

Binding of complement by complexes between antibodies to nucleic acid constituents and short oligodeoxyribonucleotides

Oligodeoxyribonucleotides act as inhibitors of the complement fixation caused by complexes between antibodies to defined oligodeoxyribonucleotides and denatured DNA. At concentrations higher than 50 micrograms oligodeoxyribonucleotide/ml complement fixation occurred in the absence of antigen. The extent of complement binding depends on the specificity of the antibodies as well as on the composition of the oligodeoxyribonucleotides. Complement fixation is observed most strongly with antisera to oligodeoxyribonucleotides and to denatured DNA, which belong predominantly to the IgM class. With two LE-sera, containing antibodies to denatured and to native DNA, no complement fixation was found. It is supposed that specific interactions of the oligodeoxyribonucleotides with amino acid residues closely neighbored to the antibody combining site lead to conformational changes in the antibody molecules and to an activation of the complement binding site.     

Binding properties of avian myeloblastosis virus DNA polymerases to nucleic acid affinity columns.

A new method for the analysis and purification of the RNA-directed DNA polymerase of RNA tumor viruses has been developed. This nucleic acid affinity chromatography system utilizes an immobilized oligo (dT) moiety annealed with poly (A). The alpha and alphabeta DNA polymerases of avain myeloblastosis virus bound effectively to poly (A) oligo (dT)-cellulose. Alpha DNA polymerase did not bind effectively to poly (A) oligo (dT)-cellulose, poly (A)-cellulose, or to cellulose. Alphabeta bound to oligo (dT)-cellulose and cellulose at the same extent (approximately 30%), indicating that this enzyme did not bind specifically to the oligo (DT) moiety only. However, alphabeta bound to poly (A)-cellulose two to three times better than to cellulose itself, showing that alphabeta could bind to poly (A) without a primer. Alphabeta DNA polymerase also bound to poly (C)-cellulose, whereas alpha did not. These data show that the alpha DNA polymerase is defective in binding to nucleic acids if the beta subunit is not present. Data is presented which demonstrates that the alphabeta DNA polymerase bound tighter to poly (A). oligo (DT)-cellulose and to calf thymus DNA-cellulose than the alpha DNA polymerase, suggesting that the beta subunit or, at least part of it is responsible for this tighter binding. In addition, alphabeta DNA polymerase is able to reversibly transcribe avian myeloblastosis virus 70S RNA approximately fivefold faster than alpha DNA polymerase in the presence of Mg2+ and equally efficient in the presence of Mn2+. alpha DNA polymerase transcribed 9S globin m RNA slightly better than alphabeta with either metal ion.