NOE Intensities Research Articles

Solution structure of the rhodanese homology domain At4g01050(175–295) from Arabidopsis thaliana

The three-dimensional structure of the rhodanese homology domain At4g01050(175-195) from Arabidopsis thaliana has been determined by solution nuclear magnetic resonance methods based on 3043 upper distance limits derived from NOE intensities measured in three-dimensional NOESY spectra. The structure shows a backbone root mean square deviation to the mean coordinates of 0.43 A for the structured residues 7-125. The fold consists of a central parallel beta-sheet with five strands in the order 1-5-4-2-3 and arranged in the conventional counterclockwise twist, and helices packing against each side of the beta-sheet. Comparison with the sequences of other proteins with a rhodanese homology domain in Arabidopsis thaliana indicated residues that could play an important role in the scaffold of the rhodanese homology domain. Finally, a three-dimensional structure comparison of the present noncatalytic rhodanese homology domain with the noncatalytic rhodanese domains of sulfurtransferases from other organisms discloses differences in the length and conformation of loops that could throw light on the role of the noncatalytic rhodanese domain in sulfurtransferases.

D(G 3T 4G 4) forms unusual dimeric G-quadruplex structure with the same general fold in the presence of K +, Na + or [formula omitted] ions

We have recently communicated that DNA oligonucleotide d(G 3T 4G 4) forms a dimeric G-quadruplex in the presence of K + ions [ J. Am. Chem. Soc. 2003, 125, 7866–7871]. The high-resolution NMR structure of d(G 3T 4G 4) 2 G-quadruplex exhibits G-quadruplex core consisting of three stacked G-quartets. The two overhanging G3 and G11 residues are located at the opposite sides of the end G-quartets and are not involved in G-quartet formation. d(G 3T 4G 4) 2 G-quadruplex represents the first bimolecular G-quadruplex where end G-quartets are spanned by diagonal (T4–T7) as well as edge-type loops (T15–T18). Three of the G-rich strands are parallel while one is anti-parallel. The G12–G22 strand demonstrates a sharp reversal in strand direction between residues G19 and G20 that is accommodated with the leap over the middle G-quartet. The reversal in strand direction is achieved without any extra intervening residues. Here we furthermore examined the influence of different monovalent cations on the folding of d(G 3T 4G 4). The resolved imino and aromatic proton resonances as well as (sequential) NOE connectivity patterns showed only minor differences in key intra- and interquartet NOE intensities in the presence of K +, Na + and NH 4 + ions, which were consistent with subtle structural differences while retaining the same folding topology of d(G 3T 4G 4) 2 G-quadruplex.

The structure and conformational behavior of sulfonium salt glycosidase inhibitors in solution: a combined quantum mechanical NMR approach.

We present the conformational analysis of an inhibitor of alpha-mannosidase, based on a novel sulfonium salt structure (1) that mimics the mannosyl cation intermediate. Because of the number of possible isomeric structures for 1, as well as its complex molecular structure, traditional conformational analysis by NMR was not applicable. Instead, a single experimentally consistent structure was obtained from finite perturbation quantum mechanical calculations of the NMR J-couplings at the B3LYP/6-311G** level. Using a full relaxation matrix analysis, we showed that the quantum-predicted NMR structure was the only isomer that was consistent with the experimental NOE intensities. The results illustrate the potential for finite perturbation calculations to be useful in the analysis of complex charged molecules.

Comparison of properties of Aib-rich peptides in crystal and solution: a molecular dynamics study.

In order to study the differences of the structural properties of Aib-rich peptides in solution and in the crystalline state, molecular dynamics (MD) simulations of the Aib-containing peptide II (pBrBz-(Aib)5-Leu-(Aib)2-OMe) were performed in the crystalline state, starting from two different conformers obtained experimentally by X-ray diffraction. The structural properties as derived from X-ray crystallography (e.g., torsional angles and hydrogen bonds) are well-reproduced in both constant-volume and constant-pressure simulations, although the force-field parameters used result in a too-high density of the crystals. Through comparison with the results from previous MD and nuclear magnetic resonance (NMR) studies of the very similar peptide I (Z-(Aib)s-Leu-(Aib)2-OMe) in dimethylsulfoxide (DMSO) solution, it is found that, in the crystal simulation, the conformational distribution of peptide II is much narrower than that in the solution simulation of peptide. I. This leads to a significant difference in 3 [symbol: see text] (HN, HC alpha) coupling constant values, in agreement with experimental data, whereas the NOE intensities or proton-proton distance bounds appear insensitive to the difference in conformational distribution. For small peptides the differences between their conformational distribution in the crystalline form and in solution may be much larger than for proteins, a fact which should be kept in mind when interpreting molecular properties in the solution state by using X-ray crystallographic data.

Effect of bulky lesions on DNA: solution structure of a DNA duplex containing a cholesterol adduct.

The three-dimensional solution structure of two DNA decamers of sequence d(CCACXGGAAC)-(GTTCCGGTGG) with a modified nucleotide containing a cholesterol derivative (X) in its C1 '(chol)alpha or C1 '(chol)beta diastereoisomer form has been determined by using NMR and restrained molecular dynamics. This DNA derivative is recognized with high efficiency by the UvrB protein, which is part of the bacterial nucleotide excision repair, and the alpha anomer is repaired more efficiently than the beta one. The structures of the two decamers have been determined from accurate distance constraints obtained from a complete relaxation matrix analysis of the NOE intensities and torsion angle constraints derived from J-coupling constants. The structures have been refined with molecular dynamics methods, including explicit solvent and applying the particle mesh Ewald method to properly evaluate the long range electrostatic interactions. These calculations converge to well defined structures whose conformation is intermediate between the A- and B-DNA families as judged by the root mean square deviation but with sugar puckerings and groove shapes corresponding to a distorted B-conformation. Both duplex adducts exhibit intercalation of the cholesterol group from the major groove of the helix and displacement of the guanine base opposite the modified nucleotide. Based on these structures and molecular dynamics calculations, we propose a tentative model for the recognition of damaged DNA substrates by the UvrB protein.

Observation of Chain Branching in Polyethylene in the Solid State and Melt via 13C NMR Spectroscopy and Melt NMR Relaxation Time Measurements

Branch contents in sparsely short-chain branched polyethylenes (<C28) and end-group concentrations in linear polyethylenes were investigated by solid- and melt-state 13C NMR using model copolymers of 1-octene, 1-octadecene, 1-hexacosene, and linear PE standards. Benchmark measurements were established for single-pulse acquisition under MAS on these melts at 423 K, using 7 mm ceramic rotors and commercially available solid-state probes at 300 and 500 MHz 1H Larmor frequency. Signal improvement by a factor of 3−4 was observed, corresponding to a factor 10 reduction in measurement time, when compared to a reference solution-state (400 MHz 1H) measurement. Transient NOE effects due to proton decoupling during FID acquisition were found to affect significantly the accuracy of branch content determination and end-group analysis in the melt spectra, even at moderate pulse repetition rates. 13C T1 and NOE buildup rates and intensity variations were established at 423 K and correlated to the local packing of the b...

Thermal unfolding of ribonuclease T1 studied by multi-dimensional NMR spectroscopy

Thermal unfolding of ribonculease (RNase) T1 was studied by 1H nuclear Overhauser enhancement spectroscopy (NOESY) and 1H- 15N heteronuclear single-quantum coherence (HSQC) NMR spectroscopy at various temperatures. Native RNase T1 is a single-chain molecule of 104 amino acid residues, and has a single alpha-helix and two beta-sheets, A and B, which consist of two and five strands, respectively. Singular value decomposition analysis based on temperature-dependent HSQC spectra revealed that the thermal unfolding of RNase T1 can be described by a two-state transition model. The midpoint temperature and the change in enthalpy were determined as 54.0 degrees C and 696 kJ/mol, respectively, which are consistent with results obtained by other methods. To analyze the transition profile in more detail, we investigated local structural changes using temperature-dependent NOE intensities. The results indicate that the helical region starts to unfold at lower temperature than some beta-strands (B3, B4, and B5 in beta-sheet B). These beta-strands correspond to the hydrophobic cluster region, which had been expected to be a folding core. This was confirmed by structure calculations using the residual NOEs observed at 56 degrees C. Thus, the two-state transition of RNase T1 appears to involve locally different conformational changes.

NMR and Monte Carlo Studies on the Solution Conformation of a Linear Capsular Polysaccharide from a Soybean‐Nodulating Bacterium (Strain B33) †

The conformational behavior of the capsular polysaccharide obtained from a fast‐growing soybean‐nodulating rhizobia (strain B33) isolated from Xinjiang Autonomous Region (Eastern China) has been analyzed by NMR and molecular mechanics simulations. This polysaccharide has the repeating unit →6)‐4‐O‐Me‐α‐d‐Glcp‐(1→4)‐3‐O‐Me‐β‐d‐GlcpA‐(1→. The NMR results indicate that the α‐(1→4) linkage may adopt a variety of conformations, and that at least two of the resulting minima must exist in solution. NOE data agree with an 85:10:5 ratio for the lowest‐energy conformations. In the case of the β‐(1→6) linkage, NMR indicates that the rotamer gg is highly populated. Experimental and calculated NOE intensities match well when the global energy minimum conformation for this linkage has exclusively the gg orientation. The influence of the adjacent methyl group on the glycosyloxymethyl population has been evaluated by simulation of a disaccharide without this group. A relative destabilization of gt rotamer has been found. Long chains have been simulated using a Metropolis algorithm at different ratios of the gg and gt rotamers in the glucose moiety. It was observed that the increase in population of the gt rotamer yielded more close contacts in the chain. †This paper is dedicated to Professor Gérard Descotes on the occasion of his 70th birthday.

Two-Dimensional NMR Study on the Structures of Micelles of Sodium Taurocholate

The aggregation behavior of sodium taurocholate (TC) in deuterium oxide without salt was investigated by one- and two-dimensional NMR spectroscopy. Analysis of the concentration dependence of the chemical shift suggests that TC forms a dimer and a pentamer. The equilibrium constants of dimerization and pentamerization are close to those already determined by chromatography in the presence of 154 mM sodium chloride. The structure of the dimer is estimated from the NOESY and ROESY spectra of a 8 mM TC solution and molecular mechanics calculations. The inter-proton distances calculated from the molecular mechanics structure are consistent with the NOE and ROE intensities, whereas those calculated from the X-ray crystal structure (hydrogen-bonded structure) are inconsistent. The molecular mechanics structure is stabilized by hydrophobic interactions between the steroid nuclei and by reduced electrostatic repulsion between the sulfonate ions. The local structures of the pentamer are estimated on the basis of t...

Conformational solution studies of [Sar7]desamino‐ and [MeAla7]desamino‐ vasopressin analogues using NMR spectroscopy

Solution structures of two analogues of vasopressin with an amino acid sequence of c[Mpa1-Tyr2-Phe3-Gln4-Asn5-Cys6]-Xaa7-Arg8-Gly9-NH2 (Xaa = Sar [I] or MeAla [II]) were established using ROE and the 3J(HNH alpha) couplings. Each of the peptides was found to exist in two stable isomers, pertaining to the cis or trans status of the Cys6-Xaa7 peptide bond, thus giving rise to four study objects. Two methods for studies of the conformational properties of the structures were compared. In the first, the algorithm consisted of three steps: (i) An Electrostatically Driven Monte-Carlo (EDMC) search for low-energy conformations. (ii) Simulations of the NOESY spectra and the vicinal couplings for these conformations. (iii) Determination of the statistical weights of the conformations with the ANALYZE package, so as to meet the best fit of the averaged NOE intensities and couplings to the experimental data. In the second method, the distance constraints and the torsion angles were used as the usual constraints in the Distance Geometry and Simulated Annealing algorithms. The flexibility of the pressin ring and the C-terminus was characterized by a large number of families of conformations. The presence of the beta-turn at position 4,5 was confirmed for all low energy structures found. The use of the EDMC method for the elaboration of the NMR data for small flexible peptides yielded an adequate description of their conformational diversity and is the method of choice for the analysis of their solution structures.

Conformation of adenosine-5′-triphosphate in the presence of Mg 2+ at different pH

NOESY experiments have been performed on adenosine-5′-triphosphate (ATP) in the presence of 1:1 Mg 2+ (MgCl 2) at different pH. The results show that H8H1′ and H8H2′ interproton NOE intensities change with pH, with nearly equal intensity at low pH (pH 1.9) and with H8H2′ signal stronger than the H8H1′ signal at higher pH (between 3.7 and 8.5). The H8H1′ and H8H2′ distances and the C4N9C1′O4′ torsion angle ( χ) have been measured from random structures of adenosine and ATP. It is concluded that the lower pH allows the formation of a P γ OMg 2+N1 bridge and the torsion angle takes a cis conformation accordingly.

Intermolecular relaxation has little effect on intra-peptide exchange-transferred NOE intensities.

Exchange-transferred nuclear Overhauser enhancement (etNOE) provides a useful method for determining the 3dimensional structure of a ligand bound to a high-molecular-weight complex. Some concern about the accuracy of such structures has arisen because indirect relaxation can occur between the ligand and macromolecule. Such indirect relaxation, or spin diffusion, would lead to errors in interproton distances used as restraints in structure determination. We address this concern by assessing the extent of intermolecular spin diffusion in nineteen peptide-protein complexes of known structure. Transferred NOE intensities were simulated with the program CORONA (Calculated OR Observed NOESY Analysis) using the rate-matrix approach to include contributions from indirect relaxation between protein-ligand and intraligand proton pairs. Intermolecular spin diffusion contributions were determined by comparing intensities calculated with protonated protein to those calculated with fully deuterated protein. The differences were found to be insignificant overall, and to diminish at short mixing times and high mole ratios of peptide to protein. Spin diffusion between the peptide ligand and the protein contributes less to the etNOE intensities and alters fewer cross peaks than the well-studied intramolecular spin diffusion effects. Errors in intraligand interproton distances due to intermolecular relaxation effects were small on average and can be accounted for with the restraint functions commonly used in NMR structure determination methods. In addition, a rate-matrix approach to calculate distances from etNOESY intensities using a volume matrix comprising only intraligand intensities was found to give accurate values. Based on these results, we conclude that structures determined from etNOESY data are no less accurate due to spin diffusion than structures determined from conventional NOE intensities.

Molecular dynamics simulations of a highly charged peptide from an SH3 domain: possible sequence-function relationship.

A seven-residue peptide that is highly conserved in SH3 domains despite being far from the active site has been shown by NMR to be stable in solution. This peptide, biologically important because it is a likely folding nucleus for SH3 domains, provides a challenging subject for molecular dynamics because it is highly charged. We present stable, 10-ns simulations of both the native-like diverging turn structure and a helical model. Free energies of these two conformations, estimated through MM-PBSA analysis using several force fields, suggest a comparable free energy (DeltaDeltaG < or =6 kcal/mol) for native and helix conformations. NOE intensities calculated from the native trajectory reproduce experimental data quite well, suggesting that the conformations sampled by the trajectory reasonably represent those observed in the NMR experiment. The molecular dynamics results, as well as sequence analysis of a diverse 690-member family of SH3 domain proteins, suggest that the presence of two elements is essential for formation of the diverging turn structure: a pair of residues with low helical propensity in the turn region and, as previously recognized, two hydrophobic residues to close the end of the diverging turn. Thus, these two sequence features may form the structural basis for the function of this peptide as a folding nucleus in this family of proteins.

Solution structure of a DNA duplex with a chiral alkyl phosphonate moiety.

The solution structures of two DNA decamers of sequence d(CCACCpxGGAAC).(GTTCCGGTGG) with a chiral alkyl phosphonate moiety (px) have been determined using NMR and restrained molecular dynamics simulations and compared with the solution structure of the unmodified duplex. The (1)H NMR spectra of two samples with pure stereochemistry in the modified phosphate have been assigned. The structures of both diastereoisomers, as well as the unmodified control duplex, have been determined from NMR-derived distance and torsion angle constraints. Accurate distance constraints were obtained from a complete relaxation matrix analysis of the NOE intensities. The structures have been refined with state of the art molecular dynamics methods, including explicit solvent and applying the particle mesh Ewald method to properly evaluate the long-range electrostatic interactions. In both cases, the calculations converge to well-defined structures, with RMSDs of approximately 1 A. The resulting structures belong to the general B family of DNA structures, even though the presence of the alkyl phosphonate moiety induces some slight displacement to the A-form in the neighborhood of the modified phosphate. Partial neutralization of this phosphate and the steric effect of the alkyl moiety provoke moderate bending in the DNA. This effect is more pronounced in the S diastereoisomer, where the alkyl group points inwards to the double helix.

The solution structure of [d(CGC)r(amamam)d(TTTGCG)]2.

The solution structure and hydration of a DNA.RNA hybrid chimeric duplex [d(CGC)r(amamam)d(TTTGCG)]2 in which the RNA adenines were substituted by 2'-O-methylated riboadenines was determined using two-dimensional NMR, simulated annealing, and restrained molecular dynamics. Only DNA residue 7T in the 2'-OMe-RNA.DNA junction adopted an O4'-endo sugar conformation, while the other DNA residues including 3C in the DNA.2'-OMe-RNA junction, adopted C1'-exo or C2'-endo conformations. The observed NOE intensity of 2'-O-methyl group to H1' proton of 4am at the DNA.2'-OMe-RNA junction is much weaker than those of 5am and 6am. The 2'-O-methyl group of 4am was found to orient towards the minor groove in the trans domain while the 2'-O-methyl groups of 5am and 6am were found to be in the gauche (+) domain. In contrast to the long-lived water molecules found close to the RNA adenine H2 and H1' protons and the methyl group of 7T in the RNA-DNA junction of [d(CGC)r(aaa)d(TTTGCG)]2, there were no long-lived water molecules found in [d(CGC)r(amamam)d(TTTGCG)]2. This is probably due to the hydrophobic enviroment created by the 2'-O-methylated riboadenines in the minor groove or due to the wider minor groove width in the middle of the structure. In addition, the 2'-O-methylation of riboadenines in pure chimeric duplex increses its melting temperature from 48.5 degrees C to 51.9 degrees C. The characteristic structural features and hydration patterns of this chimeric duplex provide a molecular basis for further therapeutic applications of DNA.RNA hybrid and chimeric duplexes with 2'-modified RNA residues.

Dimeric Solution Structure of Two Cyclic Octamers: Four-Stranded DNA Structures Stabilized by A:T:A:T and G:C:G:C Tetrads

The solution structure of two cyclic octamers of sequence d<pTGCTCGCT> and d<pCATTCATT> has been determined by two-dimensional NMR spectroscopy and restrained molecular dynamics. The two molecules dimerize at high oligonucleotide concentrations, forming a four-stranded symmetric structure. A complete relaxation matrix analysis of the NOE intensities was carried out, providing a set of around 400 accurate distance constraints. For each oligonucleotide, the calculation converges to very well-defined structures, consisting of two stacks, each with two intermolecular Watson−Crick base pairs. The central residues of each subunit are involved in the base pairs, forming two G:C:G:C or A:T:A:T tetrads, respectively. In both cases, the tetrads are formed by facing the minor groove side of the Watson−Crick base pairs. The tetrads are connected by short loops of two residues. While the residues in the second position of each loop are mainly disordered, nucleotides in the first position (thymines in both cases) are well-defined and form a cap at both ends of the stacks. This is the first time that oligonucleotide structures with minor groove aligned G:C:G:C or A:T:A:T tetrads are observed in solution. Although both oligonucleotides adopt a similar structure, the relative stability of the two molecules is different, with ΔG025 values for dimer formation of −32 and −13 kJ/mol, respectively.

Mapping the active site of angiotensin-converting enzyme by transferred NOE spectroscopy.

The interaction of five furylacryloyl (fa)-amino acid derivatives, fa-Phe, fa-Phe-Phe, fa-Gly-Leu-NH(2), fa-Ala-Lys, and fa-Trp, with angiotensin-converting enzyme (ACE), a protein of MW = 130 kDa, was studied by transferred NOESY experiments. Identification of fa derivatives binding to ACE as well as determination of their relative affinities could be accomplished directly from the compound mixtures. Of the five fa derivatives we found that fa-Phe, fa-Trp, and fa-Gly-Leu-NH(2) bind more strongly to ACE than the other two. The dissociation constant of fa-Phe was determined from NMR spectra to 5 x 10(-4) M. A large excess of dipeptides competitively displaced fa-Trp and fa-Phe-Phe from the receptor pocket, allowing the binding site to be mapped. Also, the relative affinities of the fa-Phe, fa-Ala-Lys, and fa-Gly-Leu-NH(2) changed after addition of the dipeptides with fa-Gly-Leu-NH(2) showing the strongest binding. In addition, the presence of a strong inhibitor of the S1' and S2' sites, namely captopril, resulted in the same transferred NOE intensities of fa-Phe, indicating that it binds solely to the S1 and S2 subsites. A rapid screening of binding specificity from mixtures is possible by using a large excess of ligand(s) in transferred NOE studies, even when relatively small amounts of protein are present.

Structural basis for the network of functional cooperativities in cytochrome c3 from Desulfovibrio gigas: solution structures of the oxidised and reduced states

Cytochrome c 3 is a 14 kDa tetrahaem protein that plays a central role in the bioenergetic metabolism of Desulfovibrio spp. This involves an energy transduction mechanism made possible by a complex network of functional cooperativities between redox and redox/protolytic centres (the redox-Bohr effect), which enables cytochrome c 3 to work as a proton activator. The three-dimensional structures of the oxidised and reduced Desulfovibrio gigas cytochrome c 3 in solution were solved using 2D 1H-NMR data. The reduced protein structures were calculated using INDYANA, an extended version of DYANA that allows automatic calibration of NOE data. The oxidised protein structure, which includes four paramagnetic centres, was solved using the program PARADYANA, which also includes the structural paramagnetic parameters. In this case, initial structures were used to correct the upper and lower volume restraints for paramagnetic leakage, and angle restraints derived from 13C Fermi contact shifts of haem moiety substituents were used for the axial histidine ligands. Despite the reduction of the NOE intensities by paramagnetic relaxation, the final family of structures is of similar precision and accuracy to that obtained for the reduced form. Comparison of the two structures shows that, although the global folds of the two families of structures are similar, significant localised differences occur upon change of redox state, some of which could not be detected by comparison with the X-ray structure of the oxidised state: (1) there is a redox-linked concerted rearrangement of Lys80 and Lys90 that results in the stabilisation of haem moieties II and III when both molecules are oxidised or both are reduced, in agreement with the previously measured positive redox cooperativity between these two haem moieties. This cooperativity regulates electron transfer, enabling a two-electron step adapted to the function of cytochromes c 3 as the coupling partner of hydrogenase; and (2) the movement of haem I propionate 13 towards the interior of the protein upon reduction explains the positive redox-Bohr effect, establishing the structural basis for the redox-linked proton activation mechanism necessary for energy conservation, driving ATP synthesis.

NMR structure determination of brefeldin-A, a 13-membered ring fungal metabolite

Dissolution of brefeldin-A in CDCl3 afforded two species in the ratio 13 : 1. Vicinal proton–proton coupling constants and NOE intensity enhancement measurements showed that the 13-membered ring conformation found by x-ray crystallography in the solid state appears to be preferred also in the solution-state major species. The observance of signals from a solution-state minor species (e.g. for the C-15 methyl) shows that at least one other 13-membered ring conformation exists in a small amount. While some degree of conformational flexibility for the major species cyclopentyl ring cannot be ruled out, the O-13 hydroxyl group is clearly axially disposed in the solution state as it is in the crystal. Comparison of the solution-state 13C NMR chemical shifts of the major species with those values measured by solid-state cross-polarization magic angle spinning show fairly good agreement with differences usually of 1 ppm, or less, with the exception of C-2,8,6,11,15 signals where differences were ca 2–3 ppm. Copyright © 2000 John Wiley & Sons, Ltd.

Determination of the populations and structures of multiple conformers in an ensemble from NMR data: multiple-copy refinement of nucleic acid structures using floating weights.

A new algorithm is presented for determination of structural conformers and their populations based on NMR data. Restrained Metropolis Monte Carlo simulations or restrained energy minimizations are performed for several copies of a molecule simultaneously. The calculations are restrained with dipolar relaxation rates derived from measured NOE intensities via complete relaxation matrix analysis. The novel feature of the algorithm is that the weights of individual conformers are determined in every refinement step, by the quadratic programming algorithm, in such a way that the restraint energy is minimized. Its design ensures that the calculated populations of the individual conformers are based only on experimental restraints. Presence of internally inconsistent restraints is the driving force for determination of distinct multiple conformers. The method is applied to various simulated test systems. Conformational calculations on nucleic acids are carried out using generalized helical parameters with the program DNAminiCarlo. From different mixtures of A- and B-DNA, minor fractions as low as 10% could be determined with restrained energy minimization. For B-DNA with three local conformers (C2'-endo, O4'-exo, C3'-endo), the minor O4'-exo conformer could not be reliably determined using NOE data typically measured for DNA. The other two conformers, C2'-endo and C3'-endo, could be reproduced by Metropolis Monte Carlo simulated annealing. The behavior of the algorithm in various situations is analyzed, and a number of refinement protocols are discussed. Prior to application of this algorithm to each experimental system, it is suggested that the presence of internal inconsistencies in experimental data be ascertained. In addition, because the performance of the algorithm depends on the type of conformers involved and experimental data available, it is advisable to carry out test calculations with simulated data modeling each experimental system studied.