An abasic site in DNA. Solution conformation determined by proton NMR and molecular mechanics calculations

We have determined the three-dimensional structure of a non-selfcomplementary nonanucleotide duplex which contains an abasic (apyrimidinic) site in the centre, i.e. a deoxyribose residue opposite an adenosine. The majority of the base and sugar proton resonances were assigned by NOESY, COSY and 2DQF spectra in D2O and H2O. We have measured the initial slope of buildup of NOEs in NOESY spectra at very short mixing times (25 to 50 ms), and from these were able to establish interproton distances for the central part of the duplex. We propose a different strategy for proton-proton distance determinations which takes into account the observed variations in correlation times for particular proton-proton vectors. A set of 31 measured interproton distances was incorporated into the refinement of the oligonucleotide structure by molecular mechanics calculations. Two structures were obtained which retain all aspects of a classical B DNA in which the unpaired adenine and the abasic deoxyribose lie inside the helix. We observe that the non-hydrogen bonded adenine is held well in the helix, the Tm of this base being the same as that of the A.T base pairs in the same duplex.     

Solution conformation of an oligonucleotide containing a G.G mismatch determined by nuclear magnetic resonance and molecular mechanics.

We have determined by two-dimensional nuclear magnetic resonance studies and molecular mechanics calculations the three dimensional solution structure of the non-selfcomplementary oligonucleotide, d(GAGGAGGCACG). d(CGTGCGTCCTC) in which the central base pair is G.G. This is the first structural determination of a G.G mismatch in a oligonucleotide. Two dimensional nuclear magnetic resonance spectra show that the bases of the mismatched pair are stacked into the helix and that the helix adopts a classical B-DNA form. Spectra of the exchangeable protons show that the two guanosines are base paired via their imino protons. For the non-exchangeable protons and for some of the exchangeable protons nuclear Overhauser enhancement build up curves at short mixing times have been measured. These give 84 proton-proton distances which are sensitive to the helix conformation. One of the guanosines adopts a normal anti conformation while the other is syn or close to syn. All non-terminal sugars are C2' endo. These data sets were incorporated into the refinement of the oligonucleotide structure by molecular mechanics calculations. The G.G mismatch shows a symmetrical base pairing structure. Although the mismatch is very bulky many of its features are close to that of normal B-DNA. The mismatch induces a small lateral shift in the helix axis and the sum of the helical twist above and below the mismatch is close to that of B-DNA.     

Solution conformation of salmon calcitonin in sodium dodecyl sulfate micelles as determined by two-dimensional NMR and distance geometry calculations

The 32 amino acid hormone salmon calcitonin was studied at pH 3.7 and 7.4 by two-dimensional NMR in sodium dodecyl sulfate (SDS) micelles at 310 K. The spectrum was fully assigned, and the secondary structure was obtained from nuclear Overhauser enhancement spectroscopy (NOESY), 3JHN alpha coupling constants, and slowly exchanging amide data. Three-dimensional structures consistent with NMR data were generated by using distance geometry calculations. A set of 260 interproton distances, derived from NOESY, and hydrogen-bond constraints, obtained from analysis of the amide exchange, were used. From the initial random conformations, 13 distance geometry structures with minimal violations were selected for further refinement with restrained energy minimization. In SDS, at both pHs, the main conformational feature of the hormone is an alpha-helix from Thr6 through Tyr22, thus including the amphipathic 8-22 segment and two residues of the Cys1-Cys7 N-terminal loop. The C-terminal decapeptide forms a loop folded back toward the helix. The biological significance of this conformation is discussed.     

Solution conformation of endothelin-3 by H NMR and distance geometry calculations

Endothelin-3 dissolved in 10% aqueous acetic acid was studied by nuclear magnetic resonance spectroscopy. A total of 363 distances (143 intra-residue, 108 sequential and 112 long range) was compiled from the nuclear Overhauser effect spectra and used in distance geometry calculations. The molecule assumes a compact conformation stabilized by hydrophobic interactions of the side chains. There is a helix-like structure between the residues 9–15 and an extended strand at the N-terminus. The C-terminus is in close proximity to the bicyclic ring.     

Abasic sites in duplex DNA: molecular modeling of sequence-dependent effects on conformation

Molecular modeling calculations using JUnction Minimization of Nucleic Acids (JUMNA) have been used to study sequence effects on the conformation of abasic sites within duplex DNA. We have considered lesions leading to all possible unpaired bases (X), adenine, guanine, cytosine, or thymine contained within two distinct sequence contexts, CXC and GXG. Calculations were carried out on DNA 11-mers using extensive conformational search techniques to locate the most stable abasic conformations and using Poisson-Boltzmann corrected electrostatics to account for solvation effects. The results, which are in very good agreement with available experimental data, point to strong sequence effects on both the position of the unpaired base (intra or extrahelical) and on the overall curvature induced by the abasic lesion. For CXC, unpaired purines are found to lie within the helix, while unpaired pyrimidines are either extrahelical or in equilibrium between the intra and extrahelical forms. For GXG, all unpaired bases lead to intrahelical forms, but with marked, sequence-dependent differences in induced curvature.     

Solution conformation of cytochrome c-551 from Pseudomonas stutzeri ZoBell determined by NMR.

1H NMR spectroscopy and solution structure computations have been used to examine ferrocytochrome c-551 from Pseudomonas stutzeri ZoBell (ATCC 14405). Resonance assignments are proposed for all main-chain and most side-chain protons. Stereospecific assignments were also made for some of the beta-methylene protons and valine methyl protons. Distance constraints were determined based upon nuclear Overhauser enhancements between pairs of protons. Dihedral angle constraints were determined from estimates of scalar coupling constants and intra-residue NOEs. Twenty structures were calculated by distance geometry and refined by energy minimization and simulated annealing on the basis of 1012 interproton distance and 74 torsion angle constraints. Both the main-chain and side-chain atoms are well defined except for two terminal residues, and some side-chain atoms located on the molecular surface. The average root mean squared deviation in the position for equivalent atoms between the 20 individual structures and the mean structure obtained by averaging their coordinates is 0.56 +/- 0.10 A for the main-chain atoms, and 0.95 +/- 0.09 A for all nonhydrogen atoms of residue 3 to 80 plus the heme group. The average structure was compared with an analogous protein, cytochrome c-551 from pseudomonas stutzeri. The main-chain folding patterns are very consistent, but there are some differences, some of which can be attributed to the loss of normally conserved aromatic residues in the ZoBell c-551.     

Solution structure of duplex DNA containing an extrahelical abasic site analog determined by NMR spectroscopy and molecular dynamics.

Translesional DNA synthesis past abasic sites proceeds with the preferential incorporation of dAMP opposite the lesion and, depending on the sequence context, one or two base deletions. High-resolution NMR spectroscopy and molecular dynamics simulations were used to determine the three-dimensional structure of a DNA heteroduplex containing a synthetic abasic site (tetrahydrofuran) residue positioned in a sequence that promotes one base deletions. Analysis of NMR spectra indicates that the stem region of the duplex adopts a right-handed helical structure and the glycosidic torsion angle is in anti orientation for all residues. NOE interactions establish Watson-Crick alignments for all canonical base pairs of the duplex. Measurement of distance interactions at the lesion site shows the abasic residue excluded from the helix. Restrained molecular dynamics simulations generated three-dimensional models in excellent agreement with the spectroscopic data. These structures show a regular duplex region and a slight bend at the lesion site. The tetrahydrofuran residue extrudes from the helix and is highly flexible. The model reported here, in conjunction with a previous study performed on abasic sites, explains the structural bias of one-base deletion mutations.     

Solution conformation of endothelin determined by means of 1H-NMR spectroscopy and distance geometry calculations

The structure of endothelin-1 (ET-1), an endothelial cell-derived peptide with vasoconstricting activity, was determined in an aqueous solution by means of a combination of NMR and distance geometry calculations. The resulting structure is characterized by an alpha-helical conformation in the sequence region, Lys9-Cys15. Furthermore, an extended structure and a turn structure exist in the Cys1-Ser4 and Ser5-Asp8 regions respectively, and no preferred conformation was found for the C-terminal part of the peptide which was not uniquely constrained by the NMR data. These structural elements, the alpha-helical structure in the sequence portion, Cys-X-X-X-Cys, and the extended structure in Cys-X-Cys, are homologous to those found commonly in several neurotoxic peptides.     

The disordered conformation of kappa-carrageenan in solution as determined by NMR experiments and molecular modeling

The conformation of kappa-carrageenan in solution was studied combining 1H and 13C NMR with molecular mechanics. The experimental conditions were chosen to characterize the disordered conformation of the polymer. Particular attention has been given to explore a wide range of experimental conditions as to the dependence on solvent (water and Me2SO), polymer concentration, temperature, pH, presence of a denaturing agent (guanidinium chloride), and of ions otherwise able to induce conformational order of the carrageenan chains, either in solution (I-) or in the gel state (Rb+). Two-dimensional NOE experiments were analyzed to obtain information on internuclear distances, and molecular mechanics provided the range of energetically accessible conformations. Two inter-residue topological constraints were clearly identified: their combination is rather restricting for the chain and suggests that the disordered conformation of kappa-carrageenan is characterized by an intrinsic stiffness with high values of persistent length and characteristic ratio. They also rule out any postulated interchain hydrogen bonds. In contrast, experiments on the temperature dependence of the chemical shift in Me2SO reveal the existence of two inter-residue intramolecular H-bonds which might contribute positively to the rigidity of the polymer chain. The overall picture emerging from the present results is that of a locally elongated 'loose single helix'.     

Solution conformation of the antitumor antibiotic chromomycin A3 determined by two-dimensional NMR spectroscopy

A conformational analysis and a complete assignment of the nonexchangeable proton resonances of chromomycin A3, dechromose-A chromomycin A3, and deacetylchromose-B chromomycin A3 were carried out in organic solvents. The resulting conformation in methanol has the three side chains of chromomycin A3 fully extended, away from one another and from the aglycon. In dichloromethane on the other hand, the drug was shown to adopt a highly compact conformation in which most of the 26 oxygen atoms in the molecule point out toward the solvent. The two carbohydrate side chains extend parallel to each other on the same side of the aglycon. Two intramolecular nuclear Overhauser enhancement contacts have been observed between different sugar units on these side chains, indicating close proximity for these moieties. In addition, the aliphatic side chain is folded toward the aglycon, parallel to the two oligosaccharide side chains. The overall conformation has a wedge-like shape with the two phenoxy groups exposed at the pointed edge. The presence of some exchange cross-peaks in the NOESY spectra suggests the presence of intramolecular hydrogen bonds that probably help to maintain the compact conformation. The derivatives of chromomycin A3 have qualitatively similar conformations, though their respective conformations are not as compact as the parent drug. The significance of these results is discussed in terms of a model of chromomycin A3 binding to DNA in the major groove.     

Molecular mechanics and dynamics of an abasic frameshift in DNA and comparison to NMR data

In a previous publication (Ph. Cuniasse, L.C. Sowers, R. Eritja, B. Kaplan, M.F. Goodman, J.A.H. Cognet, M. Le Bret, W. Guschlbauer and G.V. Fazakerley, Biochemistry 28, 2018 (1989), we determined by two dimensional NMR studies and molecular mechanics calculations the three-dimensional structure of a non-selfcomplementary oligonucleotide: [sequence; see text] where dr, at the center of the first strand, is a model abasic site. In order to explain all the results arising from NMR measurements, we found that an equilibrium between two conformations was necessary. These conformations differ mainly by the sugar pucker of G5 which is C2' endo or C3' endo. The latter is stabilized by addition of counterions between phosphate residues P3 and P4. In this paper, we have constructed systematically, all possible structures as a function of torsion angles delta of dr4 and of G5 by molecular mechanics in the presence or absence of counterions. Since these conformations were not forced with NMR distance measurements, this method allows detailed comparisons between all possible conformations and NMR data. Maps of contour lines of the potential energy, of fits to NMR distance measurements, and of helical twist as a function of torsion angles delta of dr4 and of G5 unravel the difficulties associated with the study of the G5 sugar pucker conformation equilibrium. Sugar puckers and proton distances are very sensitive criteria to monitor molecular dynamics. Relying on these experimental criteria, we have tested many molecular dynamics preparation phases and we propose a new warm-up and equilibration procedure for molecular dynamics. Thus we show with a 290 ps molecular dynamic run that G5 is in conformational equilibrium and that all NMR data are well reproduced.     

Solution conformation on bovine growth hormone releasing factor by 1H NMR and molecular modeling.

The structure of bovine growth hormone releasing factor (bGHRF) consisting of 44 amino acids has been studied in CD and 1H nuclear magnetic resonance (NMR) spectroscopy in conjunction with molecular modeling. Since bGHRF does not have an ordered structure in water alone, a 30% 2,2,2-trifluoroethanol (TFE) aqueous solvent was used to induce considerable alpha-helical structures, which corresponds to a helical content of approximately 62% as determined by circular dichroism (CD). The secondary structure was obtained from nuclear Overhauser enhancement and 3J(HN alpha) coupling constant in 30% TFE solution. Three-dimensional structures consistent with NMR data were generated by using distance geometry calculation. A set of 267 interproton distances derived from nuclear Overhauser effect correlation spectroscopy (NOESY) experiments and coupling constants were used. From the initial random conformations, 50 distance geometry structures with minimal violations were selected for further refinement. The 14 best structures were obtained after simulated annealing calculation with energy minimization. The structure of bGHRF in 30% TFE solution was characterized by one alpha-helix (residues 8-19), two poorly constrained helices (residues 23-27 and residues 31-34) and a beta I(III)-turn fragment (residues 20-23; phi(i+1) = -53.1 degrees, psi(i+1) = -19.6 degrees, phi(i+2) = -59.9 degrees, psi(i+2) = -20.6 degrees) connected by the segments of less defined structures in N-terminal and omega-shaped flexible C-terminal determined from NOESY cross peaks between helical segment (residues 14-18) and tail fragment (residues 42-44). The obtained structure will play an important role toward the understanding of the structural and functional role of the GHRF.     

Solution structure of the Lewis x oligosaccharide determined by NMR spectroscopy and molecular dynamics simulations.

The Lewis x (Lex) determinant is a trisaccharide fragment that has been implicated as a specific differentiation antigen, as a tumor antigen, and as a key component of the ligand for the endothelial leukocyte adhesion molecule, ELAM-1. High-resolution nuclear magnetic resonance spectroscopy shows it to have a relatively rigid structure. Only a small range of glycosidic dihedral angles in the trisaccharide produce simulated nuclear Overhauser effect spectra agreeing with data measured for the human milk pentasaccharide, lacto-N-fucopentaose-3, which contains the Lex determinant. Independently, the same average structure for the Lex determinant arises from in vacuo molecular dynamics simulations. The proposed conformation of the Lex trisaccharide is very similar to that recently determined for the closely related Lea trisaccharide. In agreement with the recent finding that both sialylated Lea and Lex react with ELAM-1, the results presented here show that the Lea and Lex determinants contain very similar carbohydrate domains.     

Solution conformation of conotoxin GI determined by 1H nuclear magnetic resonance spectroscopy and distance geometry calculations

Conformational analysis of conotoxin GI, one of the neurotoxic peptides produced by a marine snail, genus Conus, was performed by a combination of nuclear magnetic resonance spectroscopy (NMR) and distance geometry calculations. The resulting conformers on minimization of the target function were classified into two groups. The difference in the structures of the conformers is mainly due to the difference in the orientation of the side chain of the tyrosyl residue. The results show that the solution structure of conotoxin GI satisfies the conformational requirements for the biological activity of an antagonist toward nicotinic cholinergic receptors elucidated in a series of studies on alkaloids. The structure is discussed on the basis of the results of comparison of the atomic arrangements of the active sites of snake venom peptides and molecular models based on the results of secondary structure prediction.     

Solution conformation of endothelin-1 by 1H NMR, CD, and molecular modeling

The solution conformation of Endothelin-1, a recently discovered bicyclic, 21 amino acid peptide, has been examined by 1H NMR in deuterated dimethylsulphoxide and circular dichroism in aqueous and organic solvents. A total of 158 NOEs were detected, which were used as distance constraints in the distance geometry program DISGEO. Two families of structures were obtained, both characterized by a helix-like region extending from Lys9 to Cys15, but with opposite "handedness". Circular dichroism studies of the peptide in both aqueous and trifluoroethanol solutions show a negative shoulder at 224 nm, characteristic of right-handed helices. Molecular dynamics and energy minimization yielded a solution structure for this new peptide compatible with all experimental observations.     

The solution structure of the circular trinucleotide cr(GpGpGp) determined by NMR and molecular mechanics calculation.

The 3'-5' circular trinucleotide cr(GpGpGp) was studied by means of 1D and 2D high resolution NMR techniques and molecular mechanics calculations. Analysis of the J-couplings, obtained from the 1H and 13C-NMR spectra, allowed the determination of the conformation of the sugar rings and of the 'circular' phosphate backbone. In the course of the investigations it was found that the Karplus-equation most recently parametrized for the CCOP J-coupling constants could not account for the measured J(C4'P) of 11.1 Hz and a new parametrization for both HCOP and CCOP coupling constants is therefore presented. Subsequent analysis of the coupling constants yielded 'fixed' values for the torsion angles beta and delta (with beta = 178 degrees and delta = 139 degrees). The value of the latter angle corresponds to an S-type sugar conformation. The torsion angles gamma and epsilon are involved in a rapid equilibrium in which they are converted between the gauche(+) and trans and between the trans and gauche(-) domain respectively. We show that the occurrence of epsilon in the gauche(-) domain necessitates S-type sugar conformations. Given the aforementioned values for beta, gamma, delta and epsilon the ring closure constraints for the ring, formed by the phosphate backbone can only be fulfilled if alpha and zeta adopt some special values. After energy minimization with the CHARMm force field only two combinations of alpha and zeta result in energetically favourable structures, i.e. the combination alpha (t)/zeta(g-) in case gamma is in a gauche(+) and epsilon is in a trans conformation, and the combination alpha (t)/zeta (g+) for the combination gamma (t)/epsilon (g-). The results are discussed in relation to earlier findings obtained for cd(ApAp) and cr(GpGp), the latter molecule being a regulator of the synthesis of cellulose in Acetobacter xylinum.     

Sequential and parallel molecular mechanics calculations

This article describes a gradient algorithm for the computational optimization of model molecular structures, and discusses the various compromises inherent in the practical expression of the algorithm in a Fortran computer program (VULCAN) for both sequential and parallel computers. Details are given of some previously undiscussed properties of gradient algorithms; various acceleration techniques are compared: and some traps for the unwary are highlighted.     

Solution structure of alpha-conotoxin ImI determined by two-dimensional NMR spectroscopy.

The three-dimensional structure of alpha-conotoxin ImI, a potent antagonist targeting the neuronal alpha7 subtype of nicotinic acetylcholine receptor (nAChR), has been investigated by NMR spectroscopy. On the basis of 181 experimental constraints, a total of 25 converged structures were obtained. The average pairwise atomic root mean square difference is 0.40+/-0.11 A for the backbone atoms. The resulting structure indicates the presence of two successive type I beta-turns and a 310 helix for residues Cys2-Cys8 and Ala9-Arg11, respectively, and shows a significant structural similarity to that of alpha-conotoxin PnIA, which is also selective for the neuronal nAChR.