Conformational characteristics of dApdA,dApdT, dTpdA,and dTpdT from energy minimization studies

The conformational characteristics of the deoxydinucleoside monophosphates with adenine and thymine bases in all possible sequences, namely, dApdA, dApdT, dTpdA, and dTpdT have been studied using an improved set of energy parameters to calculate the total potential energy and an improved set of energy parameters to calculate the total potential energy and an improved version of the minimization technique to minimize the total energy by allowing all seven dihedral angles of the molecular fragment to vary simultaneously. The results reveal that the most preferred conformation in all these units usually corresponds to one of the four helical conformations, namely, the A-DNA, B-DNA, C-DNA, and Watson-Crick DNA models. These helical conformations differ in energies by about 3 kcal/mol with respect to one another. The conformations which could promote a loop or bend in the backbone are, in general, less stable by about 3.5 kcal/mol with respect to the respective lowest-energy helical conformation. The results indicate that there is a definite influence of bases and their actual sequences on the preferred conformations of the deoxydinucleoside monophosphates. The lowest-energy structure, although corresponding to one of the four helical conformations, differ with the type of the deoxydinucleoside monophosphate. Good or reasonable base stacking is noted in dApdA and dTpdA with both C(3′)-endo and C(2′)-endo sugars and in dApdT and dTpdT with only C(3′)-endo sugar. The inversion of the base sequence in deoxydinucleoside monophosphates alters the order of preference of low-energy conformations as well as the base-stacking property of the unit. The paths linking the starting and final states in the (ω′, ω) plane show interesting features with regard to the energy spread, thus providing insight into the path of conformational movement ofthe molecule under slight perturbation. The stabilities of the A and B forms, including the internal energies of the C(3′)-endo ans C(2′)-endo sugar systems, indicate that for dTpdT the B → A transition is less probable. For dApdA, dApdT, and dTpdA this transition is probable in the same order of preference. We propose that the T-A sequence in the polynucleotide chain might serve as the site accessible for B ? A transitions. The theoretical predictions are in good agreement with the experimental observations.     

A model for the single stranded random coil form of polydeoxyadenylic acid from minimum energy conformations of the dimeric subunit.

The minimum energy conformations of dApdA have been examined for their suitability as buildings blocks of the single stranded coil form of polynucleotides. Calculations of the characteristic ratio C difference = less than ro greater than 2/n liter2 were made for a polymer generated from all the low energy conformers, as well as for selected combinations. A polymer composed of a conformer with omega', omega = t*,g+,(skewed) psi = t, C-(2)-endo type pucker, in combination with the 'B' form, has a C difference equal to that observed in coils of apurinic acid (6) when the fraction of 'B' form conformers is approximately 25% and approximately 91%. The t*,g+ conformer is the second lowest energy form in the C-(2)-endo puckering domain, following the 'B' form.     

Conformational analysis of a covalently cross-linked Watson-Crick base pair model

Low-temperature NMR experiments and molecular modeling have been used to characterize the conformational behavior of a covalently cross-linked DNA base pair model. The data suggest that Watson-Crick or reverse Watson-Crick hydrogen bonding geometries have similar energies and can interconvert at low temperatures. This low-temperature process involves rotation about the crosslink CH(2)C(5') (psi) carbon-carbon bond, which is energetically preferred over the alternate CH(2)N(3) (phi) carbon-nitrogen bond rotation.     

Model building of DNA/RNA triple helix containing L-deoxyadenosine.

A three-dimensional model of DNA/RNA triple helix that contains a poly(L-deoxyadenosine) (L-dA) chain is proposed based on computer-assisted model building and energy calculations. The model building was performed by a new method that systematically searches possible conformations of nucleotide units in the helical chains. Two possible orientations of sugar-phosphate chains, in which two homopyrimidine strands are parallel or antiparallel with each other, were considered in the systematic search. Several possible base-pairing models, in which there are one Watson-Crick base pair and one other base pair, were also considered. Many possible models selected by the systematic search were further refined through molecular mechanics calculation incorporating a helical boundary condition. The preferred model, which was selected on the basis of potential energy, was the one with Watson-Crick and Hoogsteen base pairs and with its two polypyrimidine chains in the antiparallel orientation. The model can explain the experimental observation that poly(L-dA) forms a stable triple helix with poly(uridylic acid) (U) but not with poly(deoxythymidylic acid) (dT).     

Conformational studies of (2''-5'') polynucleotides: theoretical computations of energy, base morphology, helical structure, and duplex formation.

A detailed theoretical analysis has been carried out to probe the conformational characteristics of (2'-5') polynucleotide chains. Semi-empirical energy calculations are used to estimate the preferred torsional combinations of the monomeric repeating unit. The resulting morphology of adjacent bases and the tendency to form regular single-stranded structures are determined by standard computational procedures. The torsional preferences are in agreement with available nmr measurements on model compounds. The tendencies to adopt base stacked and intercalative geometries are markedly depressed compared to those in (3'-5') chains. Very limited families of regular monomerically repeating single-stranded (2'-5') helices are found. Base stacking, however, can be enhanced (but helix formation is at the same time depressed) in mixed puckered chains. Constrained (2'-5') duplex structures have been constructed from a search of all intervening glycosyl and sugar conformations that form geometrically feasible phosphodiester linkages. Both A- and B-type base stacking are found to generate non-standard backbone torsions and mixed glycosyl/sugar combinations. The 2'- and 5'-residues are locked in totally different arrangements and are thereby prevented from generating long helical structures.     

Energy-minimized conformation of gramicidin-like channels. I. Infinitely long poly-(L,D)-alanine beta 6.3-helix.

The energy-minimized conformation of an infinitely long poly-(L,D)-alanine in single-stranded beta 6.3-helix was calculated by the molecular mechanics method. When energy minimization was started from a wide range of initial geometries, six optimized conformations were obtained and identified as the right- and left-handed counterparts of the beta 4.5-, beta 6.3-, and beta 8.2-helices. It was found that their conformation energies increase in this order, the beta 4.5-helix having the lowest energy. The backbone dihedral angles of the energy-minimized beta 6.3-helix were: phi L = -116 degrees (or -131 degrees), psi L = 122 degrees (or 111 degrees), phi D = 131 degrees (or 116 degrees), psi D = -111 degrees (or -122 degrees), omega L = 173 degrees (or 173 degrees), and omega D = -173 degrees (or -173 degrees) for the right-handed (or left-handed) helix. This helix was composed of 6.30 residues/turn with a pitch of 4.97 A. All the alpha-carbons of L- and D-configurations appeared on one common circular helix. Interestingly, small deviations (approximately 7 degrees) of the peptide bonds from the planar structure caused a considerable lowering of the conformation energy, and, at the same time, they produced more favorable fitting of the hydrogen bonds; the carbonyl oxygens and the nearest-neighbor alpha-hydrogens also took more favorable relative positions.     

NMR structures of r(GCAGGCGUGC)2 and determinants of stability for single guanosine-guanosine base pairs

Nucleotides in RNA that are not Watson-Crick-paired form unique structures for recognition or catalysis, but determinants of these structures and their stabilities are poorly understood. A single noncanonical pair of two guanosines (G) is more stable than other noncanonical pairs and can potentially form pairing structures with two hydrogen bonds in four different ways. Here, the energetics and structure of single GG pairs are investigated in several sequence contexts by optical melting and NMR. The data for r(5'GCAGGCGUGC3')(2), in which G4 and G7 are paired, are consistent with a model in which G4 and G7 alternate syn glycosidic conformations in a two-hydrogen-bond pair. The two distinct structures are derived from nuclear Overhauser effect spectroscopic distance restraints coupled with simulated annealing using the AMBER 95 force field. In each structure, the imino and amino protons of the anti G are hydrogen bonded to the O6 and N7 acceptors of the syn G, respectively. An additional hydrogen-bond connects the syn G amino group to the 5' nonbridging pro-R(p) phosphate oxygen. The GG pair fits well into a Watson-Crick helix. In r(5'GCAGGCGUGC3')(2), the G4(anti), G7(syn) structure is preferred over G4(syn), G7(anti). For single GG pairs in other contexts, exchange processes make interpretation of spectra more difficult but the pairs are also G(syn), G(anti). Thermodynamic data for a variety of duplexes containing pairs of G, inosine, and 7-deazaguanosine flanked by GC pairs are consistent with the structural and energetic interpretations for r(5'GCAGGCGUGC3')(2), suggesting similar GG conformations.     

Conformational studies of cyclic tetrapeptides. Evidence for a bis gamma-turn conformation for chlamydocin and Ala4-chlamydocin in nonpolar solvents

The conformations of chlamydocin and cyclo (Ala-Aib-Phe-D-Pro) (Ala4-chlamydocin) in chloroform have been investigated by nuclear magnetic resonance, infrared and circular dichroism spectroscopy. The data obtained from these experiments establish an all transoid, bis gamma-turn conformation for both compounds in chloroform with the following torsional angles (+/- 20 degrees): Ala4-chlamydocin: Aib, phi + 60 degrees, psi - 50 degrees; omega + 160 degrees; Phe phi - 120 degrees, psi + 120 degrees, omega - 160 degrees; D-Pro phi + 60 degrees, psi - 55 degrees, omega + 160 degrees; Ala phi - 110 degrees, psi + 110 degrees, omega - 160 degrees. Chlamydocin adopts a closely related conformation in neat chloroform. Nuclear Overhauser Effect (NOE) data are utilized to assign amide bond geometries in the cyclic tetrapeptide ring system.     

Chemical and enzymatic probing of spatial structure of the omega leader of tobacco mosaic virus RNA

The 5′-untranslated sequence of tobacco mosaic virus RNA — the so-called omega leader — exhibits features of a translational enhancer of homologous and heterologous mRNAs. The absence of guanylic residues, the presence of multiple trinucleotide CAA repeats in its central region, and the low predictable probability of the formation of an extensive secondary structure of the Watson-Crick type were reported as the peculiarities of the primary structure of the omega leader. In this work we performed chemical and enzymatic probing of the secondary structure of the omega leader. The isolated RNA comprising omega leader sequence was subjected to partial modifications with dimethyl sulfate and diethyl pyrocarbonate and partial hydrolyses with RNase A and RNase V1. The sites and the intensities of the modifications or the cleavages were detected and measured by the primer extension inhibition technique. The data obtained have demonstrated that RNase A, which attacks internucleotide bonds at the 3′ side of pyrimidine nucleotides, and diethyl pyrocarbonate, which modifies N7 of adenines not involved in stacking interactions, weakly affected the core region of omega leader sequence enriched with CAA-repeats, this directly indicating the existence of a stable spatial structure. The significant stability of the core region structure to RNase A and diethyl pyrocarbonate was accompanied by its complete resistance against RNase V1, which cleaves a polyribonucleotide chain involved in Watson-Crick double helices and generally all A-form RNA helices, thus being an evidence in favor of a non-Watson-Crick structure. The latter was confirmed by the full susceptibility of all adenines and cytosines of the omega polynucleotide chain to dimethyl sulfate, which exclusively modifies N1 of adenines and N3 of cytosines not involved in Watson-Crick interactions. Thus, our data have confirmed that (1) the regular (CAA)_n sequence characteristic of the core region of the omega leader does form stable secondary structure, and (2) the structure formed is not the canonical double helix of the Watson-Crick type.     

Conformational analysis of levanbiose by molecular mechanics.

A relaxed conformational energy map for levanbiose, O-beta-D-fructofuranosyl-(2----6)-beta-D-fructofuranoside, was computed with the molecular mechanics program MM2(87). All torsion angles of the three linkage bonds were driven by 30 degrees increments while two primary alcohol groups were held at three staggered forms. The steric energy of all other parameters was optimized. The side groups were retained at the same relative positions on the two rings in this first part of the study so our results are directly applicable to the study of polymeric levan with identical repeating units. The low-energy dimers did not lead to viable polymers. The interresidue linkage torsion angles defined by C-6-O-2'-C-2'-C-1' (phi) and O-5-C-5-C-6-O-2' (omega) have minima at +60 degrees and -60 degrees, respectively, with accessible minima at other staggered forms. As observed in inulobiose, the preferred torsion angle at central linkage bond defined by C-5-C-6-O-2'-C-2' (psi) was antiperiplanar. An analysis of all conformations of staggered side groups showed that the C-1 and C-1' groups had little effect but the C-6' group showed a preference for chi-6'(O-5'-C-5'-C-6'-O-6') = -60 degrees. The fructofuranose rings were started at the low-energy 4(3)T conformation (angle of pseudorotation, phi 2 = 265 degrees) that was retained except when the linkage conformations created severe inter-residue conflict.     

Structural features and stability of an RNA triple helix in solution.

A 30 nt RNA with a sequence designed to form an intramolecular triple helix was analyzed by one-and two-dimensional NMR spectroscopy and UV absorption measurements. NMR data show that the RNA contains seven pyrimidine-purine-pyrimidine base triples stabilized by Watson-Crick and Hoogsteen interactions. The temperature dependence of the imino proton resonances, as well as UV absorption data, indicate that the triple helix is highly stable at acidic pH, melting in a single sharp transition centered at 62 degrees C at pH 4.3. The Watson-Crick and Hoogsteen pairings are disrupted simultaneously upon melting. The NMR data are consistent with a structural model where the Watson-Crick paired strands form an A-helix. Results of model building, guided by NMR data, suggest a possible hydrogen bond between the 2' hydroxyl proton of the Hoogsteen strand and a phosphate oxygen of the purine strand. The structural model is discussed in terms of its ability to account for some of the differences in stability reported for RNA and DNA triple helices and provides insight into features that are likely to be important in the design of RNA binding compounds.     

Predominant torsional forms adopted by dipeptide conformers in solution: parameters for molecular recognition.

The present paper describes the predominant conformational forms adopted by dipeptides in aqueous solution. More than 50 dipeptides were subjected to conformational analysis using SYBYL Random Search. The resultant collections of conformers for individual dipeptides, for small groups with related side chain residues and for large groups of about 50 dipeptides were visualized graphically and analysed using a novel three-dimensional pseudo-Ramachandran plot. The distribution of conformers, weighted according to the percentage of each in the total conformer pool, was found to be restricted to nine main combinations of backbone psi (psi) and phi (phi) torsion angles. The preferred psi values were in sectors A7 (+150 degrees to +/-180 degrees), A10 (+60 degrees to +90 degrees) and A4 (-60 degrees to -90 degrees), and these were combined with preferred phi values in sectors B12 (-150 degrees to +/-180 degrees), B9 (-60 degrees to -90 degrees) and B2 (+30 degrees to +60 degrees). These combinations of psi and phi values are distinct from those found in common secondary structures of proteins. These results show that although dipeptides can each adopt many conformations in solution, each possesses a profile of common conformers that is quantifiable. A similarly weighted distribution of dipeptide conformers according to distance between amino-terminal nitrogen and carboxyl-terminal carbon shows how the preferred combinations of backbone torsional angles result in particular N-C geometries for the conformers. This approach gives insight into the important conformational parameters of dipeptides that provide the basis for their molecular recognition as substrates by widely distributed peptide transporters. It offers a basis for the rational design of peptide-based bioactive compounds able to exploit these transporters for targeting and delivery.     

The structure of triple helical poly(U).poly(A).poly(U) studied by Raman spectroscopy

Using Raman spectroscopy, we examined the ribose-phosphate backbone conformation, the hydrogen bonding interactions, and the stacking of the bases of the poly(U).poly(A).poly(U) triple helix. We compared the Raman spectra of poly(U).poly(A).poly(U) in H2O and D2O with those obtained for single-stranded poly(A) and poly(U) and for double-stranded poly(A).poly(U). The presence of a Raman band at 863 cm-1 indicated that the backbone conformations of the two poly(U) chains are different in the triple helix. The sugar conformation of the poly(U) chain held to the poly(A) by Watson-Crick base pairing is C3' endo; that of the second poly(U) chain may be C2' endo. Raman hypochromism of the bands associated with base vibrations demonstrated that uracil residues stack to the same extent in double helical poly(A).poly(U) and in the triple-stranded structure. An increase in the Raman hypochromism of the bands associated with adenine bases indicated that the stacking of adenine residues is greater in the triple helix than in the double helical form. Our data further suggest that the environment of the carbonyls of the uracil residues is different for the different strands.     

NMR and molecular modeling studies of the interaction between wheat germ agglutinin and the beta-D-GlcpNAc-(1-->6)-alpha-D-Manp epitope present in glycoproteins of tumor cells

The beta-D-GlcpNAc-(1-->6)-alpha-D-Manp disaccharide is a constituent of highly branched cell-surface glycoconjugates that are malignancy markers. The conformational preference of the disaccharide beta-D-GlcpNAc-(1-->6)-alpha-D-Manp-OMe in solution has been studied by molecular modeling and NMR spectroscopy including 1D (1)H,(1)H T-ROESY experiments and analysis of (3)J(H,H) of the hydroxymethyl group being part of the glycosidic linkage of the disaccharide, which revealed the relative populations of the omega torsion angle as gt = 0.60, gg = 0.35, and tg = 0.05. Good agreement was obtained between the effective proton-proton distances from the experiment and those obtained by molecular modeling when the flexibility at the omega torsion angle was taken into account. Molecular modeling of the disaccharide in the binding sites of the lectin wheat germ agglutinin indicates that several conformations could be adopted in the bound state. (1)H NMR and transfer NOESY experiments confirmed that binding took place, and trans-glycosidic proton-proton interactions indicated that a conformational preference was present in the bound state, as observed by the relative change of the NOEs from H1' to H6(pro-R) and H6(pro-S). STD NMR experiments showed that binding occurred in the region of the N-acetyl group of the terminal sugar residue. In addition, the O-methyl group received saturation transfer because of the proximity to the protein. (1)H,(1)H NOEs indicated that the two methyl groups were close in space, as observed in only one of the predicted bound conformations. Experimental and theoretical data therefore agree that one conformation with a gt conformation of the hydroxymethyl group and a negative sign for the psi torsion angle is indeed selected by the lectin upon binding.     

DNA models for A, B, C and D conformations related to fiber X-ray, infrared and NMR measurements

A conformational analysis of the A, B, C and D DNA forms was made in order to establish molecular models presenting a good agreement with experimental data obtained from fiber X-ray, infrared linear dichroism and 31P NMR. The proposed models have been refined and do present good stereochemistry and optimized H-bond distances between bases associated with the Watson-Crick pairing. The DNA conformations proposed are a left handed double helix for the C form and right handed helices for A, B and D. Relations to conformational transitions between these forms are discussed.     

Helix geometry of single stranded DNA ''A'' and ''B'' forms from minimum energy conformations of dimeric subunits.

Low energy conformations with dihedral angles similar to those occurring in fibers of the 'A' and 'B' forms of DNAs have been calculated for the deoxydinucleoside phosphates dApdA, dCpdC, dTpdT, dGpdG and dGpdC (1-3). These conformers have been used as building blocks for generating larger single stranded polymers, whose helical parameters we have calculated. We find that single stranded 'A' and 'B' form helices tend to be narrower and more tightly wound than the duplexes obtained in fibers (4,5). This is consistent with experimental observations on single stranded fibers of poly (rC) (6). We also find that the different sequences have different helix geometries. In addition, it is observed that large variations in helix geometry for a given sequence are achievable at little energetic cost.     

Novel eGZ-motif formed by regularly extruded guanine bases in a left-handed Z-DNA helix as a major motif behind CGG trinucleotide repeats

The expansion of d(CGG) trinucleotide repeats (TRs) lies behind several important neurodegenerative diseases. Atypical DNA secondary structures have been shown to trigger TR expansion: their characterization is important for a molecular understanding of TR disease. CD spectroscopy experiments in the last decade have unequivocally demonstrated that CGG runs adopt a left-handed Z-DNA conformation, whose features remain uncertain because it entails accommodating GG mismatches. In order to find this missing motif, we have carried out molecular dynamics (MD) simulations to explore all the possible Z-DNA helices that potentially form after the transition from B- to Z-DNA. Such helices combine either CpG or GpC Watson-Crick steps in Z-DNA form with GG-mismatch conformations set as either intrahelical or extrahelical; and participating in BZ or ZZ junctions or in alternately extruded conformations. Characterization of the stability and structural features (especially overall left-handedness, higher-temperature and steered MD simulations) identified two novel Z-DNA helices: the most stable one displays alternately extruded Gs, and is followed by a helix with symmetrically extruded ZZ junctions. The G-extrusion favors a seamless stacking of the Watson-Crick base pairs; extruded Gs favor syn conformations and display hydrogen-bonding and stacking interactions. Such conformations could have the potential to hijack the MMR complex, thus triggering further expansion.     

Fluorescent 2''(3'')-O-aminoacylnucleosides-acceptor substrates for ribosomal peptidyltransferase+.

2'(3')-O-L-Phenylalanylderivatives of fluorescent 1,N6-ethenoadenosine and 3,N4-ethenocytidine were prepared by chemical synthesis. Both compounds are good acceptor substrates in ribosomal peptidyltransferase reactions. Since these compounds cannot form Watson-Crick base pairs, the results indicate that the terminal aminoacyladenosine unit of AA-tRNA is bound to ribosomal protein on the acceptor site of peptidyltransferase and not to rRNA.     

Chiral phosphorothioate analogues of B-DNA. The crystal structure of Rp-d[Gp(S)CpGp(S)CpGp(S)C]

The compound Rp-d[Gp(S)CpGp(S)CpGp(S)C], an analogue of the deoxyoligomer d(G-C)3, crystallizes in space group P2(1)2(1)2(1) with a = 34.90 A, b = 39.15 A and c = 20.64 A. The structure, which is not isomorphous with any previously determined deoxyoligonucleotide, was refined to an R factor of 14.5% at a resolution of 2.17 A, with 72 solvent molecules located. The two strands of the asymmetric unit form a right-handed double helix, which is a new example of a B-DNA conformation and brings to light an important and overlooked component of flexibility of the double helix. This flexibility is manifest in the alternation of the backbone conformation between two states, defined by the adjacent torsion angles epsilon and zeta, trans . gauche-(BI) and gauche-. trans (BII). BI is characteristic of classical of B-DNA and has an average C(1') to C(1') separation of 4.5 A. The corresponding separation for BII is 5.3 A. Each state is associated with a distinct phosphate orientation where the plane of the PO2 (or POS) group is alternately near horizontal or vertical with respect to the helix axis. The BI and BII conformations are out of phase on the two strands. As a consequence, on one strand purine-pyrimidine stacking is better than pyrimidine-purine, while the converse holds for the other strand. At each base-pair step, good and bad stacking alternate across the helix axis. The pattern of alternation is regular in the context of a fundamental dinucleotide repeat. Re-examination of the B-DNA dodecamer d(C-G-C-G-A-A-T-T-C-G-C-G) shows that the C-G-C-G regions contain the BI and BII conformations, and the associated dual phosphate orientation and asymmetric base stacking. Different mechanisms are used in the two structures to avoid clashes between guanine residues on opposite strands, a combination of lateral slide, tilt and helical twist in the present structure, and base roll, tilt and longitudinal slide (Calladine rules) in the dodecamer. The flexibility of the phosphate orientations demonstrated in this structure is important, since it offers a structural basis for protein-nucleic acid recognition.     

Oscillatory behaviour and unsaturated fatty acid composition of diabetic liver mitochondria

Volume oscillations of liver mitochondria resulting from valinomycin induced K+ transport, may be represented by the equation At/Am = C'.exp(-beta t).sin(omega 1t+ psi) where At is the oscillation amplitude at time t; Am, the maximal amplitude; beta, the damping coefficient, omega 1 the oscillation frequency, and C' and psi, constants. The kinetic parameters beta and omega 1 increased as a function of valinomycin concentration. Measurement of beta and omega 1 for mitochondria from normal rats (A); diabetic rats (B), and normal rats fed corn oil or lard-supplemented diets (C and D, respectively), yielded an increase in beta (P less than 0.05) in B and D as compared with A, and a decrease in omega 1 in B and D as compared with A and C, respectively. Analysis of mitochondrial lipids revealed significant diminution of arachidonic acid and other polyenoic fatty acids in diabetic and lard-fed rats, as compared with normal rats and corn oil-fed rats, respectively. The conclusion is drawn that the abnormal oscillatory behaviour of diabetic liver mitochondria is related to the alteration of the membrane fatty acid composition.