Conformations of Cyclobut-A and Cyclobut-G: structural resemblance to nucleosides and incorporation into double helical DNA.

Conformational energy calculations have been carried out on two modified nucleosides Cyclobut-A and Cyclobut-G using the methods of molecular mechanics (MM2) obtainable in the computational software MacroModel. Conformations were generated as a function of the torsion angles equivalent to the glycosidic and backbone torsions in deoxyribonucleotides. The structural resemblance of the energy minimized models of the modified nucleosides to their corresponding deoxyribonucleosides has been investigated. It is found that conformations which lie within 3 kcal/mole of the global minimum do resemble the overall shape and volume of the B-DNA nucleoside. Following this result, two deoxypentanucleotides d(GCGCG).d(CGCGC) and d(ATATA).d(TATAT) were model built incorporating cyclobut-G and cyclobut-A, respectively. These were then energy refined using the molecular mechanics package AMBER. The resultant structures demonstrate that cyclobut-A and cyclobut-G can be easily accommodated in double helical polynucleotides with minimal overall distortions.     

Conformations of Cyclobut-A and Cyclobut-G: Structural Resemblance to Nucleosides and Incorporation into Double Helical DNA

Abstract

Conformational energy calculations have been carried out on two modified nucleosides Cyclobut-A and Cyclobut-G using the methods of molecular mechanics (MM2) obtainable in the computational software MacroModel. Conformations were generated as a function of the torsion angles equivalent to the glycosidic and backbone torsions in deoxyribonucleotides. The structural resemblance of the energy minimized models of the modified nucleosides to their corresponding deoxyribonucleosides has been investigated. It is found that conformations which lie within 3 kcal/mole of the global minimum do resemble the overall shape and volume of the B-DNA nucleoside. Following this result, two deoxypentanucleotides d(GCGCG) · d(CGCGC) and d(ATATA) · d(TATAT) were model built incorporating cyclobut-G and cyclobut-A, respectively. These were then energy refined using the molecular mechanics package AMBER. The resultant structures demonstrate that cyclobut-A and cyclobut-G can be easily accommodated in double helical polynucleotides with minimal overall distortions.     

The effect of two antipodal fluorine-induced sugar puckers on the conformation and stability of the Dickerson-Drew dodecamer duplex [d(CGCGAATTCGCG)]2.

UV thermal melting studies, CD and NMR spectroscopies were employed to assess the contribution of antipodal sugar conformations on the stability of the canonical B-DNA conformation of the Dickerson-Drew dodecamer duplex [[d(CGCGAATTCGCG)]2, (ODN 1)]. Different oligodeoxynucleotide versions of ODN 1 were synthesized with modified thymidine units favoring distinct sugar conformations by using a 3'- endo (north) 2'-fluoro-2'-deoxyribofuranosyl thymine (1) or a 2'- endo (south) 2'-fluoro-2'-deoxyarabinofuranosyl thymine (2). The results showed that two south thymidines greatly stabilized the double helix, whereas two north thymidines destabilized it by inducing a more A-like conformation in the middle of the duplex. Use of combinations of north and south thymidine conformers in the same oligo destabilized the double helix even further, but without inducing a conformational change. The critical length for establishing a detectable A-like conformation in the middle of a B-DNA ODN appears to be 4 bp. Our results suggest that manipulation of the conformation of DNA in a sequence-independent manner is possible.     

Two-dimensional nuclear Overhauser enhancement investigation of the solution structure and dynamics of the DNA octamer [d(GGTATACC)]2

The resonances of nearly all 70 of the non-exchangeable protons of the duplex [d(GGTATACC)]2 in aqueous solution are assigned by proton two-dimensional nuclear Overhauser enhancement (2D NOE) spectra obtained in pure absorption phase at 500 MHz. Experimental and theoretical 2D NOE spectra are compared at each mixing time (100, 175, 250 and 400 ms) using two B-DNA structures: a standard B-form and an energy-minimized form. The GG and CC ends of the octamer duplex are well represented by the regular B-DNA structure. But large discrepancies from these models are observed for the 'TATA' box. All 2D NOE data are consistent with nanosecond correlation times, as indicated by non-selective proton spin-lattice relaxation times, but small variations in the correlation time are observed, suggesting that there are some local differences in mobility within the octamer duplex structure in solution.     

Solution structure of the EcoRI DNA octamer containing 5-fluorouracil via restrained molecular dynamics using distance and torsion angle constraints extracted from NMR spectral simulations.

The self-complementary DNA octamer [d(GGAATUFCC)]2, containing the EcoRI recognition sequence with one of the thymines replaced by 5-fluorouracil (UF), was synthesized. Proton homonuclear two-dimensional nuclear Overhauser effect (2D NOE) and double-quantum-filtered correlation (2QF-COSY) spectra, as well as one-dimensional spectra at different temperatures, were recorded for the octamer. Consequently, all proton resonances were assigned. The thermally induced transition from the duplex to single strands has been followed, demonstrating the stability of the duplex containing 5-fluorouracil. Simulations of the 2QF-COSY cross-peaks by means of the programs SPHINX and LINSHA were compared with experimental data, establishing scalar coupling constants for the sugar ring protons and hence sugar pucker parameters. The deoxyribose rings exhibit a dynamic equilibrium of N- and S-type conformers with 75-95% populations of the latter. Two programs used for complete relaxation matrix analysis 2D NOE spectra, CORMA and MARDIGRAS, were modified to account for the influence of the fluorines on dipolar interactions in the proton system. Quantitative assessment of the 2D NOE cross-peak intensities for different mixing times, in conjunction with the program MARDIGRAS, gave a set of interproton distances for each mixing time. The largest and smallest values of each of the interproton distances were chosen as the upper and lower bounds for each distance constraint. The distance bounds define the size of a flat-well potential function term, incorporated into the AMBER force field, which was employed for restrained molecular dynamics calculations. Torsion angle constraints in the form of a flat-well potential were also constructed from the analysis of the sugar pucker data. Several restrained molecular dynamics runs of 35 ps were performed, utilizing 284 experimental distance and torsion angle constraints and two different starting structures, energy-minimized A- and B-DNA. Convergence to similar structures with a root-mean-square deviation of 1.2 A was achieved for the central hexamer of the octamer, starting from A- and B-DNA. The average structure from six different molecular dynamics runs was subjected to final restrained energy minimization. The resulting final structure was in good agreement with the structures derived from different molecular dynamics runs and showed a substantial improvement of the 2D NOE sixth-root residual index in comparison with classical and energy-minimized B-DNA. A detailed analysis of the conformation of the final structure and comparison with structures of similar sequences, obtained by different methods, were performed.     

Stability and motion of a hairpin and the corresponding mismatched duplex: a theoretical exploration using molecular mechanics and normal mode analysis of 2D NMR results on d(GCCGCAGC)

The oligomer d(GCCGCAGC) can adopt two different conformations: i) a duplex with two mismatched A.C base pairs and ii) a hairpin with two C.G base pairs and a single stranded loop. We report molecular mechanics, normal mode analysis, and thermodynamic stability calculations for both structures. We show that the energy-minimized structure and harmonic-dynamics results are in complete agreement with the observed NOE spectrum and imino proton exchange data. We conclude that the high stability of the hairpin structure over the duplex at low salt concentration is due to the higher vibrational entropy contribution to the system free energy by the single stranded loop and to the lack of minor groove phosphate/phosphate electrostatic repulsions that tend to destabilize the duplex.     

Quantum-mechanical conformational analysis of the 5'-thymidilic acid molecule

The conformational analysis of the DNA structural unit--the nucleotide with thymine base and electroneutral phosphate group at 5'-position-has been carried out with the applied quantum mechanics methods at the MP2/6-311++G(d,p) // B3LYP/6-31G(d,p) theory level. As many as 660 conformations with relative Gibbs energies under standard conditions from 0 to 11.1 kcal/mole have been found. Among them, six conformations are similar to the structure of the nucleotide of AI-DNA, one--to AII- and seven--to the DNA in BI-form. The lowest Gibbs energy among the DNA-like conformations (deltaG = 2.7 kcal/mole) belongs to BI-DNA-like structure. It is shown that the glycoside chemical bond is the most labile one. The role of intramolecular CH...O hydrogen bonds in formation of the 5'-thymidilic acid molecule structure is demonstrated.     

Base-pair opening and spermine binding--B-DNA features displayed in the crystal structure of a gal operon fragment: implications for protein-DNA recognition.

A sequence that is represented frequently in functionally important sites involving protein-DNA interactions is GTG/CAC, suggesting that the trimer may play a role in regulatory processes. The 2.5 A resolution structure of d(CGGTGG)/d(CCACCG), a part of the interior operator (OI, nucleotides +44 to +49) of the gal operon, co-crystallized with spermine, is described herein. The crystal packing arrangement in this structure is unprecedented in a crystal of B-DNA, revealing a close packing of columns of stacked DNA resembling a 5-stranded twisted wire cable. The final structure contains one hexamer duplex, 17 water molecules and 1.5 spermine molecules per crystallographic asymmetric unit. The hexamer exhibits base-pair opening and shearing at T.A resulting in a novel non-Watson-Crick hydrogen-bonding scheme between adenine and thymine in the GTG region. The ability of this sequence to adopt unusual conformations in its GTG region may be a critical factor conferring sequence selectivity on the binding of Gal repressor. In addition, this is the first conclusive example of a crystal structure of spermine with native B-DNA, providing insight into the mechanics of polyamine-DNA binding, as well as possible explanations for the biological action of spermine.     

Bulge-out structures in the single-stranded trimer AUA and in the duplex (CUGGUGCGG).(CCGCCCAG). A model-building and NMR study

Model-building studies were carried out on the trimer AUA. Bulge-out structures which allow incorporation into a continuous RNA helix were generated and energy-minimized. All geometrical features obtained by previous NMR studies on purine-pyrimidine-purine sequences are accounted for in these models. One of the models was used to fit into a double helical fragment. Only minor changes were necessary to construct a central bulge-out in an otherwise intact duplex. NMR and model-building studies were performed on the duplex (CUGGUGCGG).(CCGCCCAG) which contains an unpaired uridine residue. NOE data, chemical-shift profiles and imino-proton resonances provided evidence that the extra U is bulged out of the duplex. The relatively small dispersion in 31P chemical shifts (approximately equal to 0.7 ppm) indicate the absence of t/g or g/t combinations for the phosphodiester angles zeta/alpha. An energy-minimized model of the duplex, which fits the present collection of data, is presented.     

Prediction of the native conformation of a polypeptide by a statistical-mechanical procedure. II. Average backbone structure of enkephalin

The average conformation of Met-enkephalin was determined by using an adaptive, importance-sampling Monte Carlo algorithm (SMAPPS—Statistical Mechanical Algorithm for Predicting Protein Structure). In the calculation, only the backbone dihedral angles (? and ψ) were allowed to vary; i.e., all side-chain (χ) and peptide-bond (ω) dihedral angles were kept fixed at the values corresponding to a low-energy structure of the pentapeptide. The total conformational energy for each randomly generated structure of the polypeptide was obtained by summing over the interaction energies of all pairs of nonbonded atoms of the whole molecule. The interaction energies were computed by the program ECEPP/2 (Empirical Conformational Energy Program for Peptides). Solvent effects were not included in the computation. The calculation was repeated until a total of 10 independent average conformations were established. The regions of conformational space occupied by the average structures were compared with the regions of low conditional free energy obtained by SMAPPS in the first paper of this series. Such a comparison provides an analysis of the capacity of SMAPPS to adjust the Monte Carlo search to regions of highest probability. The results demonstrate that the ability of SMAPPS to focus the Monte Carlo search is excellent. Finally, the 10 independent average conformations and the mean of the 10 average structures were utilized as the initial conformations for a direct energy minimization of the pentapeptide. Of the 11 final energy-minimized structures, three of the conformations were found to be equivalent to the conformation of lowest energy determined previously. In addition, all but two of the remaining energy-minimized structures were found to correspond to one of the two other conformations of high probability obtained in the first paper of this series. These results indicate that a set of independent average conformations can provide a rational, unbiased choice for the initial conformation, to be used in a direct energy minimization of a polypeptide. The final energy-minimized structures consequently constitute a set of low-energy conformations, which include the global energy minimum.     

Interactions that favor the native over the non-native disulfide bond among residues 58-72 in the oxidative folding of bovine pancreatic ribonuclease A

In the initial stages of the oxidative folding of both bovine pancreatic ribonuclease A (RNase A) and a 58-72 fragment thereof from the fully reduced, denatured state, the 65-72 correctly paired disulfide bond forms in preponderance over the incorrectly paired 58-65 disulfide bond. Since both disulfide-bonded loops contain the same number of amino acid residues, the question arises as to whether the native pairing results from interactions within the 58-72 segment that lead to a nativelike structure even in its fully reduced form. To answer this question, the chain buildup procedure, based on ECEPP, including a solvation treatment, was used to generate the low-energy structures for the 58-72 RNase segment, beginning with residue 72 and building back to residue 58; in this fragment, all three Cys residues (at positions 58, 65, and 72) initially exist in the reduced (CysH) state. After the open-chain energy minima of the 65-72 peptide were generated, these conformations were allowed to form the 65-72 disulfide bond, and the energies of the resulting oxidized conformations were reminimized and rehydrated. The global minimum of the loop-closed 65-72 structure and many of the low-lying loop-closed minima could be superimposed on the energy-minimized X-ray structure for residues 65-72. The low-energy structures for the full open chain 58-72 peptide were then computed and were allowed to form disulfide bonds either between residues 65 and 72 (native) or between residues 58 and 65 (non-native), and their energies were reminimized and rehydrated in the loop-closed state. Although the overall fold of the 65-72 loop-closed global minimum was the same as for the energy-minimized X-ray structure of these residues, the overall rms deviation was 3.9 A because of local deviations among residues 58-64. In contrast, the 65-72 segment of the global minimum of the 58-72 fragment could be superimposed on the corresponding residues of the energy-minimized X-ray structure. The lowest-energy structure for the 58-65 non-native paired 58-72 sequence was 6 kcal/mol higher in energy than that for the 58-72 peptide with the 65-72 disulfide bond formed. These results suggest that the native pairing of the 65-72 peptide arises from energetic determinants (adoption of left-handed single-residue conformations by Gly 68, and side chain interactions involving Gln 69) contained within this peptide sequence.     

The solution structure of a DNA duplex containing a single 1-(2-O,3-C-ethylene-beta-D-arabinofuranosyl)thymidine nucleoside

The structure of a DNA duplex containing one 1-(2-O,3-C-ethylene-beta-D-arabinofuranosyl)-thymidine nucleoside (T5) modification was investigated by use of two-dimensional 1H NMR spectroscopy at 750 MHz. The structure of the d(CCGCT5AGCG):d(CGCTAGCGG) duplex (CT5AG) containing one of this 2'-O,3'-C-linked bicycloarabino conformational restricted modification has been determined. We obtained inter-proton distance bounds from NOESY spectra by including a complete relaxation matrix analysis. These distance bounds were used as restraints in molecular dynamics (rMD) calculations. We also analyzed the fine structure of the cross peaks in a selective DQF-COSY spectra to determine the sugar conformations of the nucleotides. Forty final structures were generated for CT5AG from A-form and B-form dsDNA starting structures. The root-mean-square deviation (RMSD) of the coordinates for the forty structures of the complex was 0.92A. The structures were observed to be markedly irregular compared to canonical B-DNA, especially in terms of large variations in propeller twist and buckle. Also, lack of stacking of two bases near the modification site is observed. The sugar conformations of all the unmodified nucleotides are close to pure C2'-endo conformation. The structural feature of CT5AG was discussed in relation to the thermal stability and resistance towards exonucleolytic degradation.     

Quantitative analysis of DNA secondary structure from solvent-accessible surfaces: the B- to Z-DNA transition as a model

Solvent structure and its interactions have been suggested to play a critical role in defining the conformation of polynucleotides and other macromolecules. In this work, we attempt to quantitate solvent effects on the well-studied conformational transition between right-handed B- and left-handed Z-DNA. The solvent-accessible surfaces of the hexamer sequences d(m5CG)3, d(CG)3, d(CA)3, and d(TA)3 were calculated in their B- and Z-DNA conformations. The difference in hydration free energies between the Z and the B conformations (delta delta GH(Z-B] was determined from these surfaces to be -0.494 kcal/mol for C-5 methylated d(CG), 0.228 kcal/mol for unmethylated d(CG), 0.756 kcal/mol for d(CA)-d(TG), and 0.896 kcal/mol for d(TA) dinucleotides. These delta delta GH(Z-B) values were compared to the experimental B- to Z-DNA transition energies of -0.56 kcal/mol that we measured for C-5 methylated d(CG), 0.69-1.30 kcal/mol reported for unmethylated d(CG), 1.32-1.48 kcal/mol reported for d(CA)-d(TG), and 2.3-2.4 kcal/mol for d(TA) dinucleotides. From this comparison, we found that the calculated delta delta GH(Z-B) of these dinucleotides could account for the previous observation that the dinucleotides were ordered as d(m5CG) greater than d(CG) greater than d(CA)-d(TG) greater than d(TA) in stability as Z-DNA. Furthermore, we predicted that one of the primary reasons for the inability of d(TA) sequences to form Z-DNA results from a decrease in exposed hydrophilic surfaces of adjacent base pairs due to the C-5 methyl group of thymine; thus, d(UA) dinucleotides should be more stable as Z-DNA than the analogous d(TA) dinucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational variety and physical properties of the 1,2-dideoxyribofuranose-5-phosphate, the model DNA monomer structural unit

The results of exhaustive quantum-mechanical conformational analysis of 1,2-dideoxyribofuranose-5-phosphate molecule, the model DNA backbone structural unit, are presented. As many as 282 conformations with the relative Gibbs energies from 0 to 8.9 kcal/mole have been obtained at the MP2/cc-pVTZ // DFT B3LYP/cc-pVTZ theory level. Among them seven structures are similar to those of the DNA backbone in its AI, BI and ZII forms, while the B-DNA-like conformation has the lowest Gibbs energy (deltaG = 3.3 kcal/mole). It is shown that the relaxed force constants values for conformational parameters of all DNA-like conformations satisfy inequality K gamma > K alpha > K epsilon > K beta.     

2'5'-linked polynucleotides do form a double-stranded helical structure: a result from the energy minimization study of A2'p5'A

Experimental results on 2′5′-linked subunit systems of nucleic acids are interpreted to substantiate the view that the 2′5′-linked polynucleotides cannot form double-stranded helical structures. In order to look into this aspect of the 2′5′-linked units, as well as to make a detailed comparison between the conformational characteristics of 3′5′- and 2′5′-linked systems, we carried out an exhaustive theoretical study on A2′p5′A. The method was to compute the various terms of energy contributions to a conformational state and then to minimize the total energy, permitting all the relevant dihedral angles to adjust themselves. Four hundred thirty two probable starting conformations were considered for this treatment, but we found only 10 of them to come under low-energy states, i.e., within 5 kcal/mol energy difference with reference to the global minimum energy state. The characteristic properties of these 10 conformations were compared in detail with those previously obtained on the corresponding 3′5′-linked subunit, as well as such units with other base sequences. As a further step, a model-building study was undertaken. Using the backbone-course, base-stacking, and hydrogen-bonding possibilities of the 10 low-energy conformations of the dimer A2′p5′A, double-stranded helical structures were scrutinized for the 2′5′-linked polynucleotide. Of a few reasonable forms, a right-handed duplex structure satisfied our requirements. We describe this new duplex, making comparisons with the standard A- and B-form states of DNA. The available experimental and theoretical results on 2′5′-linked systems are also analyzed.     

Calorific Content of Certain Bacteria and Fungi

Calorific contents of dried cells of several representative species of bacteria (gram-negative rods and gram-positive rods and cocci), two species of yeasts, and a filamentous fungus were determined by bomb calorimetry. The grand mean was 5,383 cal per g of ash-free dry weight. This value was then used to determine quantity of energy assimilated (E(a)) during growth. Subsequently, E(a) was employed in the equation: Y(kcal) = Y/(E(a) + E(d)), where Y(kcal) is the yield of cells per kilocalorie of energy taken from a culture medium, Y is the yield per mole of substrate utilized, E(a) is Y times caloric content of the cells, and E(d) is the energy expended by oxidative dissimilation. An estimate of E(d) was obtained for a number of experiments by multiplying the moles of oxygen consumed per mole of substrate utilized during growth by the average quantity of energy utilized to reduce a mole of oxygen with electrons from organic compounds (106 kcal). From previous studies in our laboratories, a value for Y(kcal) of 0.118 g/kcal was predicted. The mean value for data from five studies of aerobic growth of prototrophic heterotrophs was found to be 0.111.     

Solution structure of [d(GTATATAC)]2 via restrained molecular dynamics simulations with nuclear magnetic resonance constraints derived from relaxation matrix analysis of two-dimensional nuclear Overhauser effect experiments

Two-dimensional nuclear Overhauser effect (2D NOE) spectra have been used as the experimental basis for determining the solution structure of the duplex [d(GTATATAC)]2 employing restrained molecular dynamics (rMD) simulations. The MARDIGRAS algorithm has been employed to construct a set of 233 interproton distance constraints via iterative complete relaxation matrix analysis utilizing the peak intensities from the 2D NOE spectra obtained for different mixing times and model structures. The upper and lower bounds for each of the constraints, defining size of a flat-well potential function term used in the rMD simulations, were conservatively chosen as the largest or smallest value calculated by MARDIGRAS. Three different starting models were utilized in several rMD calculations: energy-minimized A-DNA, B-DNA, and a structure containing wrinkled D-DNA in the interior. Considerable effort was made to define the appropriate force constants to be employed with the NOE terms in the AMBER force field, using as criteria the average constraints deviation, the constraints violation energy and the total energy. Of the 233 constraints, one was generated indirectly, but proved to be crucial in defining the structure: the cross-strand A5-H2 A5-H2 distance. As those two protons resonate isochronously for the self-complementary duplex, the distance cannot be determined directly. However, the general pattern of 2D NOE peak intensities, spin-lattice relaxation time (T1) values, and 31P nuclear magnetic resonance spectra lead to use of the A3-H2 A7-H2 distance for A5-H2 A5-H2 as well. Five rMD runs, with different random number seeds, were made for each of the three starting structures with the full distance constraint set. The average structure from all 15 runs and the five-structure averages from each starting structure were all quite similar. Two rMD runs for each starting structure were made with the A5-H2 A5-H2 constraint missing. The average of these six rMD runs revealed differences in structure, compared to that with the full set of constraints, primarily for the middle two base-pairs involving the missing cross-strand constraint but global deviations also were found. Conformational analysis of the resulting structures revealed that the inner four to six base-pairs differed in structure from the termini. Furthermore, an alternating structure was suggested with features alternating for the A-T and T-A steps.