Conformational properties of adenylyl-3' leads to 5'-adenosine in aqueous solution.

A detailed 220-MHz NMR study has been made of the conformational properties for the homodinucleotide adenylyl-3' leads to 5'-adenosine, ApA, in D2O. Unambiguous signal assignments of all proton signals were made with the aid of selectively deuterated nucleotidyl units, ApA, ApA, and D-8ApA, and complete, accurate sets of NMR parameters were derived by simulation-iteration methods. Sets of limiting chemical shifts and coupling values were also obtained for ApA and constituent monomers 3'-AMP and 5'-AMP at infinite dilution and at identical ionization states for assessment of dimerization effects. Conformational properties were evaluated quantitatively for most of the conformational bonds of ApA and these are consistent with two compact folded dynamically averaged structures, a base-stacked right helical structure, I, characterized as anti, C3'-endo, g-, w,w' (320,330 degrees), g'g', gg, C3'-endo, anti, and a more loosely base-stacked loop structure, II, with anti, C3'-endo, g-, w,w' (80 degrees, 50 degrees), g'g', gg, C3'-endo, anti orientations. Dimerization produces a number of nucleotidyl conformational changes including a shift in ribose equilibrium C2'-endo (S) in equilibrium C3'-endo (N) in favor of C3'-endo in both Ap- and -pA (60:40 vs. 35:65 in monomers), a change in glycosidic torsion angle chiCN toward 0 degrees, and a greater locking-in of rotamers along bonds involved in the phosphodiester backbone. Moreover, there is clear evidence that the transitions from S leads to N forms and chiCN leads to 0 degrees are directly related to base stacking in ApA. Finally, ApA exists in solution as an equilibrium between I, II and an unstacked form(s) with as yet undetermined conformational features. Since C4'-C5', C5'-O5', and C3'-O3' bonds possess exceptional conformational stabilities, it is proposed that destacking occurs primarily by rotation about P-O5' and/or O3'-P. Predominant factors influencing the overall ApA conformation are thus base-base interaction and flexibility about P-O5' and O3'-P, with change of ribose conformation occurring in consequence of an alteration of chiCN, the latter in turn being governed by the need for maximum eta overlap of stacked adenine rings.     

Significant conformational changes in an antigenic carbohydrate epitope upon binding to a monoclonal antibody.

Transferred nuclear Overhauser enhancement spectroscopy (TRNOE) was used to observe changes in a ligand's conformation upon binding to its specific antibody. The ligands studied were methyl O-beta-D-galactopyranosyl(1----6)-4-deoxy-4-fluoro-beta-D-galactopyra nos ide (me4FGal2) and its selectively deuteriated analogue, methyl O-beta-D-galactopyranosyl(1----6)-4-deoxy-2-deuterio-4-fluoro-beta -D- galactopyranoside (me4F2dGal2). The monoclonal antibody was mouse IgA X24. The solution conformation of the free ligand me4F2dGal2 was inferred from measurements of vicinal 1H-1H coupling constants, long-range 1H-13C coupling constants, and NOE cross-peak intensities. For free ligand, both galactosyl residues adopt a regular chair conformation, but the NMR spectra are incompatible with a single unique conformation of the glycosidic linkage. Analysis of 1H-1H and 1H-13C constants indicates that the major conformer has an extended conformation: phi = -120 degrees; psi = 180 degrees; and omega = 75 degrees. TRNOE measurements on me4FGal2 and me4F2dGal2 in the presence of the specific antibody indicate that the pyranose ring pucker of each galactose ring remains unchanged, but rotations about the glycosidic linkage occur upon binding to X24. Computer calculations indicate that there are two sets of torsion angles that satisfy the observed NMR constraints, namely, phi = -152 +/- 9 degrees; psi = -128 +/- 7 degrees; and omega = -158 +/- 6 degrees; and a conformer with phi = -53 +/- 6 degrees; psi = 154 +/- 10 degrees; and omega = -173 +/- 6 degrees. Neither conformation is similar to any of the observed conformations of the free disaccharide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural studies of 5-ethyl-2'-deoxyuridine by selective pulse 1H DPFGSE NOE spectroscopy and PM3 calculations.

The solution conformation of 5-ethyl-2'-deoxyuridine (EDU) has been calculated from the vicinyl proton-proton NMR coupling constants and nuclear Overhauser (NOE) distances using excitation sculpting of selective pulses (Double Pulsed Field Gradient Spin Echo NOE) at 500 MHz and molecular modelling (PM3) studies.     

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.     

Structural Studies of 5-Ethyl-2′-Deoxyuridine by Selective Pulse 1H DPFGSE NOE Spectroscopy and PM3 Calculations

Abstract

The solution conformation of 5-ethyl-2′-deoxyuridine (EDU) has been calculated from the vicinyl proton-proton NMR coupling constants and nuclear Overhauser (NOE) distances using excitation sculpting of selective pulses (Double Pulsed Field Gradient Spin Echo NOE) at 500 MHz and molecular modelling (PM3) studies.     

Combined use of homo- and heteronuclear coupling constants as restraints in molecular dynamics simulations.

A penalty function for scalar coupling constants has been applied in molecular dynamics simulations as an experimental constraint. The function is based on the difference between the coupling constant calculated from the dihedral angle and the experimentally measured coupling constant. The method is illustrated on a model cyclic pentapeptide for which 3JHN-H alpha and 3JHN-C beta, both about the phi backbone dihedral angle, have been measured. The function is efficient in producing structures consistent with the scalar couplings, but removed from the conformation observed in solution. This arises from the lack of J restraints for the psi dihedral angle. Simulations with both nuclear Overhauser effect (NOE) and J-coupling restraints illustrates small but significant differences from simulations using only NOEs.     

Nuclear Overhauser effect and cross-relaxation rate determinations of dihedral and transannular interproton distances in the decapeptide tyrocidine A.

The following interproton distances are reported for the decapeptide tyrocidine A in solution: (a) r(phi) distances between NH(i) and H alpha (i), (b) r(psi) distances between NH (i + 1) and H alpha (i), (c) r(phi psi) distances between NH(i + 1) and NH(i), (d) NH in equilibrium NH transannular distances, (e) H alpha in equilibrium H alpha transannular distances, (f) r x 1 distances between H alpha and H beta protons, (g) NH(i) in equilibrium H beta (i) distances, (h) NH (i + 1) in equilibrium H beta (i) distances, (i) carboxamide-backbone protons and carboxamide-side chain proton distances, (j) side chain proton-side chain proton distances. The procedures for distance calculations were: NOE ratios and calibration distances, sigma ratios and calibration distances, and correlation times and sigma parameters. The cross-relaxation parameters were obtained from the product, say, of NOE 1 leads to 2 and the monoselective relaxation rate of proton 2; the NOEs were measured by NOE difference spectroscopy. The data are consistent with a type I beta-turn/ type II' beta-turn/ approximately antiparallel beta-pleated sheet conformation of tyrocidine A in solution and the NOEs, cross-relaxation parameters, and interproton distances serve as distinguishing criteria for beta-turn and beta-pleated sheet conformations. It should be borne in mind that measurement of only r phi and r psi distances for a decapeptide only defines the ( phi, psi)-space in terms of 4(10) possible conformations; the distances b-j served to reduce the degeneracy in possible (phi, psi)-space to one tyrocidine A conformation. The latter conformation is consistent with that derived from scalar coupling constants, hydrogen bonding studies, and proton-chromophore distance measurement, and closely resembles the conformation of gramicidin S.     

Molecular orbital studies on nucleoside analogs. II. Conformation of 6-azapyrimidine nucleosides.

PCILO (Perturbative Configuration Interaction using Localised Orbitals) computations have been carried out for three 6-azapyrimidine nucleosides, 6-azauridine, 6-azacytidine and 6-azathymidine, for both C(2')-endo and C(3')-endo pucker of the sugar ring. The results indicate a syn (chiCN=180 degrees) conformation followed by chiCN=90 degrees and gg conformation for C(3')-endo 6-aza analogs as compareed to the anti (chiCN=0 degrees) and gg conformation preferred by the corresponding pyrimidine nucleosides. For C(2')-endo sugar geometry, 6-azauridine and 6-azacytidine prefer, respectively, chiCN=0 degrees (anti) and phi C(4')-C(5')=60 degrees C (gg) and chiCN-240 degrees (syn) and phi C(4')-C(5')=120 degrees. The corresponding nucleosides, uridine and cytidine, show a preference for syn (chiCN=240 degrees) and gg and anti(chiCN=0 degrees) and gg , respectively. The X-ray crystallographic conformations of 6-azauridine and 6-azacytidine have been attributed to intermolecular hydrogen bonding and crystal packing forces. The results of PMR, CD and ORD studies on 6-azauridine and 6-azacytidine in aqueous solutions are in agreement with the PCILO predictions.     

Stabilization of RNA stacking by pseudouridine.

The effect of the modified nucleoside pseudouridine (psi) on RNA structure was compared with uridine. The extent of base stacking in model RNA oligonucleotides was measured by 1H NMR, UV, and CD spectroscopy. The UV and CD results indicate that the model single-stranded oligoribonucleotides AAUA and AA psi A form stacked structures in solution and the CD results for AA psi A are consistent with a general A-form helical conformation. The AA psi A oligomer exhibits a greater degree of UV hypochromicity over the temperature range 5-55 degrees C, consistent with a better stacked, more A-form structure compared with AAUA. The extent of stacking for each nucleotide residue was inferred from the percent 3'-endo sugar conformation as indicated by the H1'-H2' NMR scalar coupling. This indirect indication of stacking was confirmed by sequential NOE experiments. NMR measurements as a function of temperature indicate that pseudouridine forms a more stable base stacking arrangement than uridine, an effect that is propagated throughout the helix to stabilize stacking of neighboring purine nucleosides. The N1-H imino proton in AA psi A exchanges slowly with solvent, suggesting a role for the extra imino proton in stabilizing the conformation of pseudouridine. These results show that the conformational stabilization is an intrinsic property of pseudouridine occurring at the nucleotide level. The characteristics of pseudouridine in these models are consistent with earlier studies on intact rRNA, indicating that pseudouridine probably performs the same stabilizing function in most structural contexts.     

Deoxyribose ring conformation of [d(GGTATACC)]2: an analysis of vicinal proton-proton coupling constants from two-dimensional proton nuclear magnetic resonance

Exchangeable and nonexchangeable protons of [d(GGTATACC)]2 in aqueous cacodylate solution were assigned from two-dimensional nuclear Overhausser effect (2D NOE) spectra. With phase-sensitive COSY and double quantum filtered COSY (DQF-COSY) experiments, the cross-peaks resulting from deoxyribose ring conformation sensitive proton-proton vicinal couplings, i.e., all 1'-2', 1'-2", 2'-3', and 3'-4' couplings and six from 2"-3' couplings, were observed. From the cross-peak fine structure, the 2',2" proton assignments can be confirmed; coupling constants J1'2' and J1'2" and sums of coupling constants involving H2' and H2" for all residues and H3' for C8 were obtained. The DISCO procedure [Kessler, H., Muller, A., & Oschkinat, H. (1985) Magn. Reson. Chem. 23, 844-852] was used to extract individual 1'-2' and 1'-2" coupling constants. The sum of coupling constants involving H1' or H3' was measured from the one-dimensional spectrum where signal overlap is not a problem. Analysis of the resulting coupling constants and sums of coupling constants, in the manner of Rinkel and Altona [Rinkel, L. J., & Altona, C. (1987) J. Biomol. Struct. Dyn. 4, 621-649], led to the following conclusion: C2'-endo deoxyribose ring conformation is predominant for every residue, but a significant amount of C3'-endo conformation may exist, ranging from 14% to 30%.     

Three-dimensional structural analysis of the group B polysaccharide of Neisseria meningitidis 6275 by two-dimensional NMR: the polysaccharide is suggested to exist in helical conformations in solution

The solution conformations of the group B polysaccharide of Neisseria meningitidis were analyzed by DQF-COSY and pure absorption 2D NOE NMR with three mixing times. The pyranose ring of the sialic acid residue was found to be in the 2C5 conformation. The DQF-COSY analysis indicated that the orientations of H6 and H7 and of H7 and H8 are both gauche. In order to overcome the difficulties in analyzing the NOE data due to the two sets of proton overlaps, molecular modeling of alpha-2,8-linked sialic acid oligomers was carried out to investigate possible conformers, and theoretical NOE calculations were performed by using CORMA (complete relaxation matrix analysis). Our analysis suggests that the polysaccharide adopts helical structures for which the phi (defined by O6-C2-O8-C8) and psi (C2-O8-C8-C7) angles are in the following ranges: phi -60 to 0 degrees, psi 115-175 degrees or phi 90-120 degrees, psi 55-175 degrees. The weak affinity of anti-B antibodies for smaller alpha-2,8-linked oligosaccharides may be due to the fact that such oligomers are more flexible and may not form an ordered structure as the poly(sialic acid) does.     

Empirical torsional potential functions from protein structure data. Phi- and psi-potentials for non-glycyl amino acid residues.

The torsional potential functions Vt(phi) and Vt(psi) around single bonds N--C alpha and C alpha--C, which can be used in conformational studies of oligopeptides, polypeptides and proteins, have been derived, using crystal structure data of 22 globular proteins, fitting the observed distribution in the (phi, psi)-plane with the value of Vtot(phi, psi), using the Boltzmann distribution. The averaged torsional potential functions, obtained from various amino acid residues in L-configuration, are Vt(phi) = 1.0 cos (phi + 60 degrees); Vt(psi) = 0.5 cos (psi + 60 degrees) - 1.0 cos (2 psi + 30 degrees) - 0.5 cos (3 psi + 30 degrees). The dipeptide energy maps Vtot(phi, psi) obtained using these functions, instead of the normally accepted torsional functions, were found to explain various observations, such as the absence of the left-handed alpha helix and the C7 conformation, and the relatively high density of points near the line psi = 0 degrees. These functions derived from observational data on protein structures, will, it is hoped, explain various previously unexplained facts in polypeptide conformation.     

A conformational study of alpha-L-Rhap-(1----2)-alpha-L-Rhap-(1----OMe) by NMR nuclear Overhauser effect spectroscopy (NOESY) and molecular dynamics calculations.

The conformational preference of the disaccharide alpha-L-Rhap-(1----2)-alpha-L-Rhap-(1----OMe) (1) about the glycosidic torsion angles, phi and psi, was studied by NMR NOESY spectroscopy and molecular mechanics calculations. The NOE data were consistent with either of two distinct conformations close to minima on a calculated phi/psi potential energy surface. Starting from the lowest energy conformation, a 1-ns molecular dynamics (MD) trajectory was computed in vacuo, from which the NOE curves were simulated and compared to the experimentally observed NOESY data.     

Design of peptides: synthesis, crystal structure, molecular conformation, and conformational calculations of N-Boc-L-Phe-Dehydro-Ala-OCH3.

It is noteworthy that the dehydro-Ala residue adopts an extended conformation that is different than those observed in dehydro-Phe, dehydro-Leu, and dehydro-Abu. The peptide N-Boc-L-Phe-dehydro-Ala-OCH3 (C18H24N2O5) was synthesized by the usual workup procedure and finally by converting N-Boc-L-Phe-L-Ser-OCH3 to N-Boc-L-Phe-dehydro-Ala- OCH3. It was crystallized from its solution in a methanol-water mixture at room temperature. The crystals belong to the monoclonic space group P2(1), with a = 9.577(1) A, b = 5.195(3) A, c = 19.563(3) A, beta = 94.67(5) degrees, V = 970.1(6) A3, Z = 2, dm = 1.201(5) Mg m-3, dc = 1.197(5) Mg m-3. The structure was determined using direct method procedures. It was refined by a full-matrix least-squares procedure to an R value of 0.048 for 1370 observed reflections. The C2 alpha-C2 beta distance is 1.327(8) A, while the bond angles N2-C2 alpha-C2' and C1'-N2-C2 alpha are 109.8(5) degrees and 127.8(5) degrees, respectively. The backbone adopts a nonspecific conformation with dehydro-Ala in a fully extended conformation with the following torsion angles: theta 1 = 175.2(4) degrees, omega 0 = 170.2(4) degrees, phi 1 = 135.8(5) degrees, psi 1 = -22.6(6) degrees, omega 1 = 168.5(5) degrees, phi 2 = -170.3(5) degrees, psi 2T = -178.6(5) degrees, theta T = 178.4(7) degrees. The rigid planar and trans conformation of dehydro-Ala forces Phe to adopt a strained conformation. The Boc group has a trans-trans conformation.(ABSTRACT TRUNCATED AT 250 WORDS)     

The crystal structure of the alpha-cellobiose.2 NaI.2 H(2)O complex in the context of related structures and conformational analysis

The crystal structure of beta-D-glucopyranosyl-(1-->4)-alpha-D-glucopyranose (alpha-cellobiose) in a complex with water and NaI was determined with Mo K(alpha) radiation at 150 K to R=0.027. The space group is P2(1) and unit cell dimensions are a=9.0188, b=12.2536, c=10.9016 A, beta=97.162 degrees. There are no direct hydrogen bonds among cellobiose molecules, and the usual intramolecular hydrogen bond between O-3 and O-5' is replaced by a bridge involving Na+, O-3, O-5', and O-6'. Both Na+ have sixfold coordination. One I(-) accepts six donor hydroxyl groups and three C-H***I(-) hydrogen bonds. The other accepts three hydroxyls, one Na+, and five C-H***I(-) hydrogen bonds. Linkage torsion angles phi(O-5) and psi(C-5) are -73.6 and -105.3 degrees, respectively (phi(H)=47.1 degrees and psi(H)=14.6 degrees ), probably induced by the Na+ bridge. This conformation is in a separate cluster in phi,psi space from most similar linkages. Both C-6-O-H and C-6'-O-H are gg, while the C-6'-O-H groups from molecules not in the cluster have gt conformations. Hybrid molecular mechanics/quantum mechanics calculations show <1.2 kcal/mol strain for any of the small-molecule structures. Extrapolation of the NaI cellobiose geometry to a cellulose molecule gives a left-handed helix with 2.9 residues per turn. The energy map and small-molecule crystal structures imply that cellulose helices having 2.5 and 3.0 residues per turn are left-handed.     

Relation between the spectral NMR parameters and conformation characteristics of the amino acid residue backbone. Analysis of elements of the secondary structure of proteins

Based on the analysis of the proton-proton distance dependences from the conformational characteristics of the L-amino acid residues, the correlation diagram of the NOE cross peak intensity waited values with the regions of the sterically allowed space (phi, psi) was proposed. The method for determining the dihedral angles phi, psi values using the information about NOE cross peak intensities was elaborated. By the model spectral NMR parameters of the bovine pancreatic trypsin inhibitor, it is shown that the accuracy of the angles phi, psi determination exceed the corresponding accuracy provided by other methods of the structural interpretation of the two-dimensional NMR spectroscopy data. The analysis of the waited spectral NMR parameters for the different types of protein regular secondary structures and beta-turns was performed.     

NMR structures of a nonapeptide from DNA binding domain of human polymerase-alpha determined by iterative complete-relaxation-matrix approach.

Nuclear magnetic resonance structures of a nonapeptide, ERFKCPCPT, selected from the DNA binding domain of human polymerase-alpha, were determined by complete relaxation matrix analysis of transverse NOE data. The structures exhibit a type III turn with residues KCPC, and the remaining residues exhibit non-ordered structures. The turn was confirmed by alpha, N (i,i+3) connectivity, a low temperature coefficient of NH chemical shift (-3.1 x 10(-3)) of the fourth residue, 3J(NHalpha) coupling constants, and characteristic CD peaks at 228 and 200 nm. Furthermore, phi and psi dihedral angles for the i + 1, and i + 2 residues of the turn are found to be -80 and -41 and -60 and -40 degrees. The first proline residue is trans- while the second exists in both cis- and trans- configurations, with trans- being more than 80% populated. The trans-configuration was established from C5alpha-P6alpha correlation and phi and psi angles of the proline. The five-membered proline ring is in DOWN puckered (C-beta-exo/C-gamma-endo) conformation. The structure of the peptide reveals that the two cysteine thiols are approximately 5 A(o) apart and appropriately positioned to covalently bind cis-diamminedichloroplatinum(II), a widely used anti-cancer drug.     

Role of azaamino acid residue in beta-turn formation and stability in designed peptide.

The structural perturbation induced by C(alpha)-->N(alpha) exchange in azaamino acid-containing peptides was predicted by ab initio calculation of the 6-31G* and 3-21G* levels. The global energy-minimum conformations for model compounds, For-azaXaa-NH2 (Xaa=Gly, Ala, Leu) appeared to be the beta-turn motif with a dihedral angle of phi= +/- 90 degrees, psi=0 degrees. This suggests that incorporation of the azaXaa residue into the i+2 position of designed peptides could stabilize the beta-turn structure. The model azaLeu-containing peptide, Boc-Phe-azaLeu-Ala-OMe, which is predicted to adopt a beta-turn conformation was designed and synthesized in order to experimentally elucidate the role of the azaamino acid residue. Its structural preference in organic solvents was investigated using 1H NMR, molecular modelling and IR spectroscopy. The temperature coefficients of amide protons, the characteristic NOE patterns, the restrained molecular dynamics simulation and IR spectroscopy defined the dihedral angles [ (phi i+1, psi i+1) (phi i+2, psi i+2)] of the Phe-azaLeu fragment in the model peptide, Boc-Phe-azaLeu-Ala-OMe, as [(-59 degrees, 127 degrees) (107 degrees, -4 degrees)]. This solution conformation supports a betaII-turn structural preference in azaLeu-containing peptides as predicted by the quantum chemical calculation. Therefore, intercalation of the azaamino acid residue into the i+2 position in synthetic peptides is expected to provide a stable beta-turn formation, and this could be utilized in the design of new peptidomimetics adopting a beta-turn scaffold.     

500-MHz 1H NMR study of poly(dG).poly(dC) in solution using one-dimensional nuclear Overhauser effect

Secondary structures of poly(dG).poly(dC) and poly(dG).poly(dm5C) in solution are determined by nuclear Overhauser effect (NOE) measurements on GH8-deuterated and -nondeuterated DNAs with low presaturation pulse lengths (10-25 ms) and low-power and prolonged accumulations in the range of 50,000-72,000 scans. Under these conditions, the NOE difference spectra were free from diffusion. Primary NOEs between base protons GH8/CH6 and sugar protons H1', H2'/H2', and H3' suggest that in poly(dG).poly(dC) both guanine and cytosine nucleotides adopt a C3'-endo, low anti X = 200-220 degrees conformation. Computer modeling of the NOE data enable identification for the first time, in terms of the geometry of the nucleotide repeat, handedness, and helix geometry, of the structure of poly(dG).poly(dC) to be the A form, and the derived structure for the polymer duplex is very close to the single crystal structure of the double-helical d-GGGGCCCC [McCall, M., Brown, T., & Kennard, O. (1985) J. Mol. Biol. 183, 385-396]. Similar nuclear Overhauser effect data on poly(dG).poly(dm5C) revealed that G and m5C adopt a C2'endo, anti X = 240-260 degrees conformation, which indicates that this DNA exhibits the B form in solution. In summary, the results presented in this paper demonstrate that methylation of cytosines in poly(dG).poly(dC) causes A----B transition in the molecule.     

Structure and solution conformations of a cyclic trisaccharide from high-resolution n.m.r. spectroscopy and molecular modelling.

The conformational properties of a cyclic trisaccharide: [O-beta-D-glucopyranosyl-(1----6)]3 1,6"-anhydride nonacetate (C36H48O24, 1) have been established by high-resolution 1H- and 13C-n.m.r. spectroscopy in conjunction with potential-energy and molecular-mechanics calculations. The n.m.r. parameters used were nuclear Overhauser enhancements (n.O.e.) and coupling constants. From theoretical models of the trisaccharide, a statistical-mechanics approach was used to compute an ensemble average-relaxation matrix from which the n.O.e. were calculated. The observed nuclear Overhauser enhancements as measured by n.m.r. spectroscopy may be satisfactorily modelled if averaging over two conformational states is considered. In solution, both conformations of the molecule exhibit three-fold symmetry; the beta-linked glucopyranose rings have the 4C1 conformation. In one conformer, the orientation about the (1----6) linkage is characterized by torsion angles phi = 79.5 degrees, psi = 143.5, and omega = -64.3. For the other conformer, these values are phi = -137.7, psi = 68.2, and omega = 45.6. The existence of such a conformer shows that solution behaviour is not dominated by the stabilizing influence of the exoanomeric effect.