The solution conformation of sialyl-α(2→6)-lactose studied by modern NMR techniques and Monte Carlo simulations

SummaryWe present a comprehensive strategy for detailed characterization of the solution conformations of oligosaccharides by NMR spectroscopy and force-field calculations. Our experimental strategy generates a number of interglycosidic spatial constraints that is sufficiently large to allow us to determine glycosidic linkage conformations with a precision heretofore unachievable. In addition to the commonly used {1H,1H} NOE contacts between aliphatic protons, our constraints are: (a) homonuclear NOEs of hydroxyl protons in H2O to other protons in the oligosaccharide, (b) heteronuclear {1H,13C} NOEs, (c) isotope effects of O1H/O2H hydroxyl groups on13C chemical shifts, and (d) long-range heteronuclear scalar coupling across glycosidic bonds.We have used this approach to study the trisaccharide sialyl-α(2→6)-lactose in aqueous solution. The experimentally determined geometrical constraints were compared to results obtained from force-field calculations based on Metropolis Monte Carlo simulations. The molecule was found to exist in 2 families of conformers. The preferred conformations of the α(2→6)-linkage of the trisaccharide are best described by an equilibrium of 2 conformers with Φ angles at −60° or 180° and of the 3 staggered rotamers of the Ω angle with a predominantgt conformer. Three intramolecular hydrogen bonds, involving the hydroxyl protons on C8 and C7 of the sialic acid residue and on C3 of the reducing-end glucose residue, contribute significantly to the conformational stability of the trisaccharide in aqueous solution.

[1]  J. Brady,et al.  A revised potential-energy surface for molecular mechanics studies of carbohydrates. , 1988, Carbohydrate research.

[2]  R. Freeman,et al.  Selective excitation at two arbitrary frequencies. The double-DANTE sequence , 1989 .

[3]  L. Mueller.  Sensitivity enhanced detection of weak nuclei using heteronuclear multiple quantum coherence , 1979 .

[4]  G. A. Jeffrey,et al.  The refinement of the crystal structures of -D-glucose and cellobiose , 1968 .

[5]  L. Poppe,et al.  Solution conformation of sialosylcerebroside (GM4) and its NeuAc(α2→3)Galβ sugar component , 1989 .

[6]  Bernd Meyer,et al.  Further justification for the exo-anomeric effect. Conformational analysis based on nuclear magnetic resonance spectroscopy of oligosaccharides , 1982 .

[7]  R. Dwek,et al.  Solution conformations of N-linked oligosaccharides. , 1987, Biochemistry.

[8]  K. Bock.  The preferred conformation of oligosaccharides in solution inferred from high resolution NMR data and hard sphere exo-anomeric calculations , 1983 .

[9]  L. Poppe,et al.  1H-detected measurements of long-range heteronuclear coupling constants. Application to a trisaccharide , 1991 .

[10]  L. Poppe,et al.  Conformation of the glycolipid globoside head group in various solvents and in the micelle-bound state , 1990 .

[11]  L. Poppe,et al.  Sequence determination in oligosaccharides by relayed NOE experiments in the rotating frame. , 1989, Biochemical and biophysical research communications.

[12]  D. Cumming,et al.  Reevaluation of rotamer populations for 1,6 linkages: reconciliation with potential energy calculations. , 1987, Biochemistry.

[13]  R. Bryant,et al.  Self-diffusion of water at the protein surface: a measurement , 1984 .

[14]  R. Jernigan,et al.  Solvent effect on binding thermodynamics of biopolymers , 1990, Biopolymers.

[15]  K. Wüthrich.  NMR of proteins and nucleic acids , 1988 .

[16]  N. Metropolis,et al.  Equation of State Calculations by Fast Computing Machines , 1953, Resonance.

[17]  A. Bax,et al.  Application of new, high-sensitivity, 1H-13C-n.m.r.-spectral techniques to the study of oligosaccharides. , 1987, Carbohydrate research.

[18]  I. Tvaroška.  Dependence on saccharide conformation of the one-bond and three-bond carbonproton coupling constants , 1990 .

[19]  D. Canet,et al.  DANTE-Z. A new approach for accurate frequency selectivity using hard pulses , 1989 .

[20]  Peter J. Smith,et al.  Polysaccharide conformation. Part VIII. Test of energy functions by Monte Carlo calculations for monosaccharides , 1975 .

[21]  M. Karplus.  Contact Electron‐Spin Coupling of Nuclear Magnetic Moments , 1959 .

[22]  H. Wennerström.  Nuclear magnetic relaxation induced by chemical exchange , 1972 .

[23]  G. Gallo,et al.  Hydrogen bonding and conformation of glucose and polyglucoses in dimethyl-sulphoxide solution , 1966 .

[24]  J F Vliegenthart,et al.  Conformational analysis of the sialyl alpha(2----3/6)N-acetyllactosamine structural element occurring in glycoproteins, by two-dimensional NOE 1H-NMR spectroscopy in combination with energy calculations by hard-sphere exo-anomeric and molecular mechanics force-field with hydrogen-bonding potential. , 1989, European journal of biochemistry.

[25]  T. Frenkiel,et al.  Long-range carbon-proton coupling constants: application to conformational studies of oligosaccharides. , 1988, Carbohydrate research.

[26]  Oligosaccharide conformations: Application of NMR and energy calculations , 1990 .

[27]  K. Wüthrich,et al.  Efficient purging scheme for proton-detected heteronuclear two-dimensional NMR , 1988 .

[28]  A. Bax,et al.  Generation of Pure Phase NMR Subspectra for Measurement of Homonuclear Coupling Constants , 1987 .

[29]  The consistent force field: Representation of molecular structure by potential energy functions , 1983 .

[30]  K. Bock,et al.  The conformations of oligosaccharides related to the ABH and Lewis human blood group determinants , 1980 .

[31]  W. Richards,et al.  Conformational transitions in N-linked oligosaccharides. , 1986, Biochemistry.

[32]  R. Lemieux.  Rhône-Poulenc Lecture. The origin of the specificity in the recognition of oligosaccharides by proteins , 1989 .

[33]  I. Tvaroška,et al.  An attempt to derive a new Karplus-type equation of vicinal proton-carbon coupling constants for COCH segments of bonded atoms , 1989 .

[34]  A. Bax.  Correction of cross-peak intensities in 2D spin-locked NOE spectroscopy for offset and Hartmann-Hahn effects , 1988 .

[35]  R. Shukla,et al.  Spin-locked states of homonuclear two-spin systems , 1988 .

[36]  C. Bauvy,et al.  Structural analysis of five lactose-containing oligosaccharides by improved, high-resolution, two-dimensional 1H-n.m.r. spectroscopy☆ , 1989 .

[37]  David P. Landau,et al.  Computer Simulation Studies in Condensed Matter Physics , 1988 .

[38]  J. Prestegard,et al.  Orientational analysis of micelle-associated trehalose using an NMR-pseudoenergy approach , 1989 .

[39]  D. G. Davis,et al.  Practical aspects of two-dimensional transverse NOE spectroscopy , 1985 .

[40]  K. Wüthrich,et al.  Application of phase sensitive two-dimensional correlated spectroscopy (COSY) for measurements of 1H-1H spin-spin coupling constants in proteins. , 1983, Biochemical and biophysical research communications.

[41]  Conformational aspects of oligosaccharides , 1990 .

[42]  L. Poppe,et al.  13C‐selective, 1H‐detected 2D {1H, 13C} correlation spectra of oligosaccharides , 1991 .

[43]  John L. Markley,et al.  Analysis of laboratory-frame and rotating-frame cross-relaxation buildup rates from macromolecules , 1989 .

[44]  J. Tropp.  Dipolar relaxation and nuclear Overhauser effects in nonrigid molecules: The effect of fluctuating internuclear distances , 1980 .

[45]  J. Feigon,et al.  Simplification of DNA proton nuclear magnetic resonance spectra by homonuclear Hartmann-Hahn edited two-dimensional nuclear overhauser enhancement spectroscopy , 1990 .

[46]  D. Davies,et al.  Intramolecular hydrogen bonding in 1'-sucrose derivatives determined by SIMPLE proton NMR spectroscopy. , 1986, Journal of the American Chemical Society.

[47]  B. Farmer,et al.  Relay artifacts in ROESY spectra , 1987 .

[48]  M. Murcko,et al.  Rotational barriers: 4. Dimethoxymethane. The anomeric effect revisited , 1989 .

[49]  D. I. Hoult,et al.  Critical factors in the design of sensitive high resolution nuclear magnetic resonance spectrometers , 1975, Proceedings of the Royal Society of London. A. Mathematical and Physical Sciences.

[50]  U. Singh,et al.  500-picosecond molecular dynamics in water of the Man alpha 1----2Man alpha glycosidic linkage present in Asn-linked oligomannose-type structures on glycoproteins. , 1990, Biochemistry.

[51]  B. Hills.  Multinuclear NMR studies of water in solutions of simple carbohydrates. , 1991 .

[52]  Y. Nishida,et al.  Preferred conformation about the C5-C6 bond of N-acetylneuraminyl(2-6)-D-galacto- and -D-glucopyranosides in solution , 1991 .

[53]  Richard R. Ernst,et al.  Coherence transfer by isotropic mixing: Application to proton correlation spectroscopy , 1983 .

[54]  L. Poppe,et al.  Nuclear magnetic resonance of hydroxyl and amido, protons of oligosaccharides in aqueous solution: evidence for a strong intramolecular hydrogen bond in sialic acid residues , 1991 .

[55]  R. Schauer.  Chemistry, metabolism, and biological functions of sialic acids. , 1982, Advances in carbohydrate chemistry and biochemistry.

[56]  John W. Brady,et al.  Molecular dynamics simulations of .alpha.-D-glucose in aqueous solution , 1989 .

[57]  Relayed-NOE experiments in the rotating frame for sequence analysis of peptides , 1988 .

[58]  E. Brown,et al.  Cell-surface carbohydrates and their interactions. I. NMR or N-acetyl neuraminic acid. , 1975, Biochimica et biophysica acta.

[59]  L. Poppe,et al.  Selective, inverse-detected measurements of long-range 13C, 1H coupling constants. Application to a disaccharide , 1991 .

[60]  A. Imberty,et al.  Conformational Analysis of Oligosaccharides: Reconciliation of Theory with Experiment , 1990 .

[61]  Homans Sw,et al.  A molecular mechanical force field for the conformational analysis of oligosaccharides: comparison of theoretical and crystal structures of Man alpha 1-3Man beta 1-4GlcNAc. , 1990 .

[62]  Domenico Acquotti,et al.  Three-dimensional structure of the oligosaccharide chain of GM1 ganglioside revealed by a distance-mapping procedure: a rotating and laboratory frame nuclear overhauser enhancement investigation of native glycolipid in dimethyl sulfoxide and in water-dodecylphosphocholine solutions , 1990 .

[63]  A. French.  Rigid‐ and relaxed‐residue conformational analyses of cellobiose using the computer program mm2 , 1988 .

[64]  Hydroxyl and amido groups as long-range sensors in conformational analysis by nuclear Overhauser enhancement: a source of experimental evidence for conformational flexibility of oligosaccharides , 1989 .

[65]  C. Bush,et al.  Molecular dynamics simulations and the conformational mobility of blood group oligosaccharides , 1990, Biopolymers.

[66]  R. Freeman,et al.  Selective excitation with the DANTE sequence. The baseline syndrome , 1989 .

[67]  F. D. Leeuw,et al.  The relationship between proton-proton NMR coupling constants and substituent electronegativities—I : An empirical generalization of the karplus equation , 1980 .

[68]  K. Koike,et al.  Three-dimensional structure of the oligosaccharide terminus of globotriaosylceramide and isoglobotriaosylceramide in solution. A rotating-frame NOE study using hydroxyl groups as long-range sensors in conformational analysis by 1H-NMR spectroscopy. , 1990, European journal of biochemistry.

[69]  H. Hori,et al.  1H-NMR Studies on (6R)- and (6S)-deuterated (1–6)-linked disaccharides: assignment of the preferred rotamers about C5–C6 bond of (1–6)-disaccharides in solution , 1985 .

[70]  James H. Prestegard,et al.  A molecular mechanics‐NMR pseudoenergy approach to the solution conformation of glycolipids , 1988 .

[71]  E. Berman.  Structural and conformational analysis of sialyloligosaccharides using carbon-13 nuclear magnetic resonance spectroscopy. , 1984, Biochemistry.

[72]  B. Meyer,et al.  A new force-field program for the calculation of glycopeptides and its application to a heptacosapeptide-decasaccharide of immunoglobulin G1. Importance of 1-6-glycosidic linkages in carbohydrate.peptide interactions. , 1990, European journal of biochemistry.