Carbohydrate structure and serological behaviour of "antifreeze" glycoproteins from an Antarctic fish.

The carbohydrate moiety of the “antifreeze” glycoprotein from $\text{Trematomus borchgrevinki}$ was found to be β-D-galactosyl 1–3 N-acetyl galactosamine by gasliquid chromatography. The glycoprotein inhibited anti-T antibody from human serum and $\text{Arachis hypogoea}$ lectin, but was inactive against $\text{Vicia graminea}$. Native “antifreeze” glycoprotein did not inhibit the agglutinins from $\text{Helix pomatia}$ or $\text{Cepaea hortensis}$, although after Smith degradation showed a strong inhibition towards them. Inhibition of the latter agglutinin demonstrates the carbohydrate-protein linkage to be α-linked. The presence of the Thomsen-Friedenreich antigen (T-antigen) on the “antifreeze” glycoprotein and its relation to tumour cell surfaces is briefly discussed.     

Conformational Analysis of the β-amyloid Peptide Fragment, β(12–28)

Abstract

NMR and CD spectroscopy have been used to examine the conformation of the peptide, β(12–28), (VHHQKLVFFAEDVGSNK) in aqueous and 60% TFE/40% H₂0 solution at pH 2.4. In 60% TFE solution, the peptide is helical as confirmed by the CD spectrum and by the pattern of the NOE cross peaks detected in the NOESY spectrum of the peptide. In aqueous solution, the peptide adopts a more extended and flexible conformation. Broadening of resonances at low temperature, temperature-dependent changes in the chemical shifts of several of the CH_α resonances and the observation of a number of NOE contacts between the hydrophobic side-chain protons of the peptide are indicative of aggregation in aqueous solution. The behavior of β(12–28) in 60% TFE and in aqueous solution are consistent with the overall conformation and aggregation behavior reported for the larger peptide fragment, β(1–28) and the parent β-amyloid peptide.     

Tyrosine optical activity as a probe of the conformation and interactions of fibronectin

A very intense negative band is observed at ～ 183 nm in the CD spectrum of fibronectin from bovine plasma. This transition has not previously been reported, probably because it occurs in a spectral region that has not been readily accessible in earlier studies. At longer wavelength, the observed CD is very similar to spectra reported for human and chick material, having positive bands at ～230 and ～200 nm, and a negative band at ～215nm. The low molar ellipticity of the negative band ([θ] ≈ ?2.5 × 10³ deg cm² dmol^?1) suggests little α-helix or β-sheet structure. The new transition, and the two positive bands at higher wavelength, do not correspond to known transitions of the peptide backbone, but all three are present in the CD of N-acetyltyrosineamide. It is therefore suggested that the observed CD behavior of fibronectin arises predominantly from the optical activity of tyrosine side chains. The contribution of this side-chain optical activity to the CD of other proteins is discussed. On raising pH to ionize tyrosine residues, the positive CD band at ～230 nm is lost in both N-acetyltyrosineamide and in fibronectin. The spectral change is fully reversible in the model compound, but only partially reversible in fibronectin. From this evidence, and the magnitude of the 183-nm band, it is suggested that some or all of the tyrosine residues in fibronectin may be present within ordered domains. The possible role of S? S bonds in maintaining tertiary structure is discussed. The interaction of fibronectin with heparin is accompanied by a large increase in the 183-nm band and by slight enhancement of the negative band at 215 nm, consistent with some limited formation of β-sheet. Present results indicate that CD may be of considerable value in characterization of the molecular organization and biologically relevant interactions of fibronectins and of related glycoproteins of the extracellular matrix.     

Circular dichroism eigenspectra of polyproline II and β-strand conformers of trialanine in water: Singular value decomposition analysis

Despite that a number of experimental and theoretical investigations have been carried out to determine the structure of trialanine in water, the reported populations of polyproline II (PPII) and β-strand conformers vary and were found to be dependent on which spectroscopic method was used. Such discrepancies are due to limitations of different spectroscopic methods used. Here, the temperature- and pH-dependent circular dichroism (CD) and NMR experiments have been carried out to develop a self-consistent singular value decomposition procedure. The temperature-dependent CD spectra indicate the presence of two conformers, but due to the two peptide bonds in a trialanine, one should take into consideration of four different conformers to fully interpret the NMR results. From the pH-dependent NMR coupling constant measurements, the conformation of zwitterionic trialanine is little different from that of cationic one. The strong pH dependency of CD spectrum is likely due to charge transfer transitions between carboxylate and nearby peptide groups or internal field effects not to pH-dependent conformational change. To simultaneously analyze the temperature-dependent CD and NMR data, a self-consistent procedure was used to newly determine the reference NMR coupling constants required to estimate one of the peptide dihedral angles. From the estimated enthalpy and entropy changes associated with the transition from enthalpically favorable PPII conformer to entropically favorable β-strand conformer, the relative populations of the four possible conformers of trialanine were determined and compared with the previous experimental findings. We anticipate that the present experimental results and interpretation procedure would be of use in determining the solution structures of small oligopeptides in the future.     

Studies on proline-containing tetrapeptide models of beta-turns

The synthesis of a series of protected tetrapeptides of the general formula Cbz-Gly-X²-Y³-Gly-OR (R = stearyl or methyl, X and/or Y = proline) is described. Detailed CD studies have been performed to evaluate the contribution of proline-containing β-turns to the CD spectra of proteins. The CD spectra of all the models are dominated by the chiral contribution of the proline residue. In polar, proton-donating solvents, a poly-proline II-like spectrum was observed in almost all cases. The tetrapeptide model Cbz-Gly-Gly-Pro-Gly-OStearyl, in acetonitrile shows a type C spectrum that has not been previously reported for linear peptides. The ir and nmr data on this model support the assumption of that of a type III β-turn, exhibiting a type C spectrum, participate in the conformational equilibrium. The most interesting finding of the CD studies is the observation of a type D spectrum (according to the classification of Woody [Woody, R. W. (1974) in Peptides, Polypeptides and Proteins, Blout, E. R. Bovey, F. A. Lotan, N. & Goodman, M. (Eds.), Wiley, New York]) for models Cbz-Gly-Pro-Asp(OBu^t)-Gly-OStearyl and Cbz-Gly-Pro-Ser(OBu^t)-Gly-OStearyl in cyclohexane. The results of the CD measurements ae discussed in correlation with ir and nmr data and with recent literature.     

Infrared study of trifluoroacetic acid unpurified synthetic peptides in aqueous solution: trifluoroacetic acid removal and band assignment

Synthetic peptide or protein samples are mostly unpurified with trifluoroacetic acid (TFA) used during the synthesis procedure, which strongly interferes with structure determination by infrared (IR) spectroscopy. The aim of this work was to propose a simple strategy to remove TFA contribution from attenuated total reflection (ATR)–IR spectra of the hexahistidine peptide (His6) in aqueous solution to study the conformation of this synthetic peptide without previous purification. Such a strategy is based on the subtraction mode widely employed to remove water contribution, and it is tested with TFA unpurified histidine as a model system. The subtraction is based on eliminating the strong TFA bands at 1147 and 1200 cm⁻¹ by applying a scaling factor (as in buffer correction). The proposed modes represent excellent strategies that do not modify spectral features, and they provide reliable routines to obtain the synthetic peptide spectrum without TFA contribution. The conformational information from the corrected spectra at different pH values is deduced from semiempirical calculated IR spectra of different His6 conformers. The spectral features and the band positions of the corrected spectrum suggest that the peptide molecules mainly adopt an intermolecular β-sheet structure.     

920 MHz ultra-high field NMR approaches to structural glycobiology

Although NMR spectroscopy has great potential to provide us with detailed structural information on oligosaccharides and glycoconjugates, the carbohydrate NMR analyses have been hampered by the severe spectral overlapping and the insufficiency of the conformational restraints. Recently, ultra-high field NMR spectrometers have become available for applications to structural analyses of biological macromolecules. Here we demonstrate that ultra-high fields offer not only increases in sensitivity and chemical shift dispersion but also potential benefits for providing unique information on chemical exchange and relaxation, by displaying NMR spectral data of oligosaccharide, glycoprotein, and glycolipid systems recorded at a 21.6 T magnetic field (corresponding to 920 MHz ¹H observation frequency). The ultra-high field NMR spectroscopy combined with sugar library and stable-isotope labeling approaches will open new horizons in structural glycobiology.     

Conformational Analysis of the Type II and Type III Collagen α-1 Chain C-Telopeptides by 1H NMR and Circular Dichroism Spectroscopy

Abstract

The type II and type III collagen α-1 chain C-telopeptides are a 27 mer with the sequence NAc- GPGIDMSAFAGLGPREKGPDPLQYMRA and a 22mer, NAc-GGGVASLGAGEKGPVG- YGYEYR, respectively. Their conformations have been studied in CD₃OH/H₂O (80/20) solution by means of two-dimensional proton NMR and CD spectroscopy. Based on TOCSY and NOESY experiments, all resonances were assigned and the conformational properties were analyzed in terms of vicinal NH-H_α coupling constants, sequential and medium range NOEs and amide proton temperature coefficients.

The conformation of the type II C-telopeptide is essentially extended. Evidence from CD spectroscopy suggests that a very minor proportion of the peptide might be helical (ca. 8%), but the NMR data show no evidence for a non-linear structure. The observation of reduced amide proton temperature dependence coefficients in certain sections of the molecule can, in view of the absence of any other supporting evidence, only be interpreted in terms of local shielding from solvent for sterical reasons (large hydrophobic side-chains).

The conformation of the type III C-telopeptide is mostly extended except for a β-turn ranging from Gly⁸ to Glu¹¹, which is stabilized by a hydrogen-bond between NH of Glu¹¹ and the carbonyl group of Gly⁸. The low temperature coefficient of NH(Glu¹¹) and, in particular, the observation of a medium range NOE between H_α (A⁹) and NH(E¹¹) corroborate the existence of a β-turn in this region. Although spectral overlap prevents a precise conclusion with regard to the type of β-turn present, there is some evidence that it might be type II.     

Synthesis and conformational analysis of N-glycopeptides. II. CD,molecular dynamics,and nmr spectroscopic studies on linear N-glycopeptides

The comprehensive structural analysis reported herein of eight N-glycopeptides, in three different solvents, is based on quantitative CD experiments, homonuclear nuclear Overhauser effect measurements, and molecular dynamics (MD) calculations. Although several orientations of the two amide planes attached to the carbohydrate pyranose ring are possible, according to NOE, CD data, and MD simulations, of all of the glycopeptide models, regardless of the type of the carrier peptide, only one dominant conformer population was found. This conformer is characterized by a nearly trans orientation of the CH and NH hydrogens of both acetamido groups. This finding is in perfect agreement with x-ray crystallographic data on the solid state conformation of the 1-N-acetyl- and 1-N-(β-aspartoyl)-2-acetamido-2-deoxy-β-D -glucopyranosylamine. The precise identification of this dominant conformer of N-glycopeptides in solution was the major question addressed herein by the structural analyses. A “CD additivity” experiment was carried out using an equimolar solution of Boc-Pro-Asp-NHCH₃ and l-N-acetyl-3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-β-D -gluco-pyranosylamine at ambient temperature in acetonitrile. The CD spectrum obtained from the equimolar solution of the above two molecules (the “spectroscopic sum”) was identical with the CD curve obtained from the algebraic summation of the individually recorded CD spectra of the peptide and the carbohydrate moiety (“mathematical sum”). The global picture of the CD spectral analyses of the eight parent peptides with the eight N-glycopeptides revealed that in trifluoroethanol and acetonitrile, the side-chain modification of the Asn models (natural N-glycopeptide analogues) by N-glycosylation has a significant effect on the conformation of the carrier peptide, resulting in a decrease in the original type I β-turn content. Simultaneously, the type II β-turn conformational percentage increased to ≈ 20%. Such a conformational ratio change seems to be larger than the expected errors arising from the CD analyses, and agrees with the results of MD calculations. N-glycosylation of Asn residues causes perturbations, not only through the covalent bond, but also through specific hydrogen bonds between the backbone and side chain atoms. CD spectroscopy, augmented by efficient CD curve deconvolution techniques, has proved to be a useful tool for studying multicomponent conformer mixtures of small linear peptides in solution and changes of conformational equilibria caused by N-glycosylation. © 1993 John Wiley & Sons, Inc.     

Conformational Studies on Calcium Binding By tBoc-Leu-Pro-Tyr-Ala-NHCH3, a Tyrosine Kinase Substrate,in a Nonpolar Solvent

Abstract

With a view to understanding the structural requirement for tyrosine phosphorylation, we have examined the free and Ca²⁺-bound conformations of the synthetic peptide tBoc-Leu-Pro-Tyr-Ala-NHCH₃, a substrate for a protein tyrosine kinase, using circular dichroism (CD), ¹H and ¹³C nuclear magnetic resonance (NMR) and molecular modeling methods. CD spectrum of the free peptide in water showed a random coil structure, while the spectrum in acetonitrile was indicative of a folded structure containing a type III β-turn. Dihedral angle data derived from J_NH-CH coupling constants, as well as two-dimensional ¹H-COSY and NOESY spectral analyses, showed that the peptide adopts a conformation close to the 3_10- helix. Ca²⁺ binding by the peptide, as monitored by CD spectral changes, was quite weak in water. However, substantial CD spectral changes were observed in the peptide on addition of Ca²⁺ in acetonitrile suggestive of major conformational alterations due to Ca²⁺ binding. Analysis of the binding isotherms at 25°C obtained from CD data in acetonitrile indicated a 2:1 peptide:Ca²⁺ (“sandwich”) complex to be the dominant species with a K_d of about 30μM. A. 1:1 complex was also present and became significant at Ca²⁺:peptide ratios above 1. By comparison, the peptide formed a predominantly 1:1 complex with Mg²⁺ with a K_d of about 40μM. ¹³C-NMR data showed that a mixture of cis and trans conformers (arising from rotation around the Leu-Pro bond) in the free peptide changes over to the all-trans form on coordination of the peptide carbonyl groups to the Ca²⁺ ion. ¹H-NOESY data of the Ca²⁺ complex revealed several interactions involving the sidechains of two peptide molecules in the sandwich. Molecular modeling and energy minimization with and without the input of NOESY-derived distance constraints showed the sandwich complex to be an energetically very favourable conformation. Besides its relevance in terms of the possible involvement of divalent cations in substrate-tyrosine kinase interaction, the conformational characterization of tBoc-Leu-Pro-Tyr-Ala-NHCH₃ and its Ca²⁺ complex should help understand the conformational determinants for Ca²⁺-binding by linear peptides.     

Assignment of the 270 MHz nuclear magnetic resonance spectrum of somatostatin in water

The proton nuclear magnetic resonance spectrum of the 14 residue peptide hormone somatostatin in D₂O at 270 MHz has been assigned by comparing the spectra of synthetic analogs with those of the native peptide. Extensive difference double resonance studies of all somatostatins and pH titrations confirmed all assignments. ³J_NHCH values and conventional NMR hydrogen bonding studies confirm the existence of preferred secondary conformations but not with a predominant conformation possessing a β-turn in either of the sequences 7–8–9–10 or 8–9–10–11. More extensive data treatment is needed before the actual conformation(s) of somatostatin is elucidated, but several NMR criteria for conformations are proposed.     

Conformational Analysis of the β-amyloid Peptide Fragment, β(12-28)

Abstract NMR and CD spectroscopy have been used to examine the conformation of the peptide, β(12-28), (VHHQKLVFFAEDVGSNK) in aqueous and 60% TFE/40% H(2)0 solution at pH 2.4. In 60% TFE solution, the peptide is helical as confirmed by the CD spectrum and by the pattern of the NOE cross peaks detected in the NOESY spectrum of the peptide. In aqueous solution, the peptide adopts a more extended and flexible conformation. Broadening of resonances at low temperature, temperature-dependent changes in the chemical shifts of several of the CH(α) resonances and the observation of a number of NOE contacts between the hydrophobic side-chain protons of the peptide are indicative of aggregation in aqueous solution. The behavior of β(12-28) in 60% TFE and in aqueous solution are consistent with the overall conformation and aggregation behavior reported for the larger peptide fragment, β(1-28) and the parent β-amyloid peptide.     

Preparation and conformational analysis of C-glycosyl β- and β/β-peptides

Ten C-glycosyl β²- and β/β²-peptides with three to eight amino acid residues have been prepared. Solution and solid-phase peptide syntheses were employed to assemble β²-amino acids in which C-glycosylic substituents are attached to the C-2 position of β-amino acids. Conformational analysis of the C-glycosyl β²-peptides using NMR and CD spectra indicates that the tripeptide can form a helical secondary structure. Besides, helix directions of the C-glycosyl β/β²-peptides are governed by the configuration at the α-carbon of the peptide backbone that originates from the stereocenter of the C-glycosyl β²-amino acids.     

Solution Structures,Dynamics, and Ice Growth Inhibitory Activity of Peptide Fragments Derived from an Antarctic Yeast Protein

Exotic functions of antifreeze proteins (AFP) and antifreeze glycopeptides (AFGP) have recently been attracted with much interest to develop them as commercial products. AFPs and AFGPs inhibit ice crystal growth by lowering the water freezing point without changing the water melting point. Our group isolated the Antarctic yeast Glaciozyma antarctica that expresses antifreeze protein to assist it in its survival mechanism at sub-zero temperatures. The protein is unique and novel, indicated by its low sequence homology compared to those of other AFPs. We explore the structure-function relationship of G. antarctica AFP using various approaches ranging from protein structure prediction, peptide design and antifreeze activity assays, nuclear magnetic resonance (NMR) studies and molecular dynamics simulation. The predicted secondary structure of G. antarctica AFP shows several α-helices, assumed to be responsible for its antifreeze activity. We designed several peptide fragments derived from the amino acid sequences of α-helical regions of the parent AFP and they also showed substantial antifreeze activities, below that of the original AFP. The relationship between peptide structure and activity was explored by NMR spectroscopy and molecular dynamics simulation. NMR results show that the antifreeze activity of the peptides correlates with their helicity and geometrical straightforwardness. Furthermore, molecular dynamics simulation also suggests that the activity of the designed peptides can be explained in terms of the structural rigidity/flexibility, i.e., the most active peptide demonstrates higher structural stability, lower flexibility than that of the other peptides with lower activities, and of lower rigidity. This report represents the first detailed report of downsizing a yeast AFP into its peptide fragments with measurable antifreeze activities.     

Copper(II) binding to two novel histidine-containing model hexapeptides: evidence for a metal ion driven turn conformation

The solution conformation and the copper(II) binding properties have comparatively been investigated for the two novel hexapeptides Ac-HPSGHA-NH₂ (P2) and Ac-HGSPHA-NH₂ (P4). The study has been carried out by means of CD, NMR, EPR and UV-Vis spectroscopic techniques in addition to potentiometric measurements to determine the stability constants of the different copper(II) complex species formed in the pH range 3-11. The peptides contain two histidine residues as anchor sites for the metal ion and differ only for the exchanged position of the proline residue with glycine. CD and NMR results for the uncomplexed peptide ligands suggest a predominantly unstructured peptide chain in aqueous solution. Potentiometric and spectroscopic data (UV-Vis, CD and EPR) show that both peptides strongly interact with copper(II) ions by forming complexes with identical stoichiometries but different structures. Furthermore, Far-UV CD experiments indicate that the conformation of the peptides is dramatically affected following copper(II) complexation with the P4 peptide adopting a β-turn-like conformation.     

Influenza virus: An NMR study of mechanisms involved in infection

High resolution ¹H-NMR spectroscopy has been used to study the infection of chicken embryo fibroblasts by influenza virus. Marked changes in the NMR spectrum occur when infectious influenza virus is introduced into the fibroblasts and these changes appear to depend upon the presence of active neuraminidase (EC 3.2.1.18). A crude preparation of neuraminidase from Vibrio cholerae is able to effect similar changes. Only minor spectral changes are observed in the absence of culture medium or when the viral genome material is inactivated by β-propiolactone. Similarly, little change is seen in the NMR spectrum when amantadine, which is thought to inhibit uncoating of the virus inside the cell, or actinomycin D, which inhibits cellular nucleic acid metabolism, are incubated with fibroblasts prior to the addition of virus. The results suggest that neuraminidase, in co-operation with a factor in the infectious process, initiates a cellular event which can be monitored by NMR. The nature of this cellular mechanism is unknown, but further studies are under way to determine its importance in viral infection.     

Secondary Structural Studies of Bovine Caseins: Structure and Temperature Dependence of β-Casein Phosphopeptide (1-25) as Analyzed by Circular Dichroism, FTIR Spectroscopy, and Analytical Ultracentrifugation

The defining structural feature of all of the caseins is their common phosphorylation sequence. In milk, these phosphoserine residues combine with inorganic calcium and phosphate to form colloidal complexes. In addition, nutritional benefits have been ascribed to the phosphopeptides from casein. To obtain a molecular basis for the functional, chemical, and biochemical properties of these casein peptides, the secondary structure of the phosphopeptide of bovine β-casein (1–25) was reexamined using Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopies. Both methods predict secondary structures for the peptide which include polyproline II elements as well as β-extended sheet and turn-like elements. These structural elements were highly stable from 5° to 70°C. Reexamination of previously published ¹H NMR data using chemical shift indices suggests structures in accord with the CD and FTIR data. Dephosphorylation showed little or no secondary structural changes, as monitored by CD and FTIR, but the modified peptide demonstrated pronounced self-association. The polymers formed were not highly temperature sensitive, but were pressure sensitive as judged by analytical ultracentrifugation at selected rotor speeds. Molecular dynamics (MD) simulations demonstrated relatively large volume changes for the dephosphorylated peptide, in accord with the pressure dependent aggregation observed in the analytical ultracentrifuge data. In contrast the native peptide in MD remained relatively rigid. The physical properties of the peptide suggest how phosphorylation can alter its biochemical and physiological properties.     

The Val-210-Ile pathogenic Creutzfeldt–Jakob disease mutation increases both the helical and aggregation propensities of a sequence corresponding to helix-3 of PrPC

A peptide corresponding to the third helical region within the PrP^C protein, from residues 198 to 218 (helix-3), was synthesised with and without the familial 210-Val to Ile Creutzfeldt–Jakob disease mutation. The NMR structure of PrP^C predicts no global variation in stability for this mutation, indicating that local sequence rather than global structural factors are involved in the pathological effects of this mutation. ¹H NMR analysis of peptides with and without this mutation indicated that it had no significant effect on local helical structure. Temperature denaturation studies monitored by CD showed that the mutation increased the helical content within this region (helical propensity), but did not stabilise the helix toward denaturation (helical stability). Aggregation data indicated that, in addition to increasing helical propensity, this mutation increased the aggregation propensity of this sequence. CD and NMR data indicate that helical interactions, stabilised by the Val-210-Ile mutation, may precede the formation of β-sheet aggregates in this peptide sequence. Therefore, this pathological mutation probably does not facilitate PrP^C to PrP^Sc conversion by directly destabilising the helical structure of PrP^C, but may preferentially stabilise PrP^Sc by facilitating β-sheet formation within this sequence region of PrP. In addition, helical interactions between helix-3 in two or more PrP^C molecules may promote conversion to PrP^Sc.     

The Principal Neutralizing Determinant of HIV-1IIIB: Conformation of the Peptide Bound to a Neutralizing Antibody Studied by 2D-NMR

Summary RP135 is a 24-residue peptide corresponding to the principal neutralizing determinant of the envelope glycoprotein gp120 of the human immunodeficiency virus type 1. We have studied the conformation of RP135 in complex with a neutralizing antibody 0.5β raised against gp120 by 2D NMR spectroscopy. The antigenic determinant recognized by this antibody was mapped using a combination of HOHAHA and ROESY measurements, in which resonances of the Fab and the tightly bound peptide residues are eliminated and the mobile residues of the bound peptide are sequentially assigned. We found that residues Ser⁶-Thr¹⁹ are part of the epitope, while Lys⁵ and Ile²⁰ are at its boundaries. Difference spectroscopy was applied to study the conformation of the bound peptide representing the epitope within the 52 kDa of the Fab complex. Specific residues of the peptide were deuterated or replaced and the difference between the NOESY spectrum of the complex with the unlabeled residue and the NOESY spectrum of the complex with the modified residue revealed the interactions of the labeled residue both within the peptide and with the Fab fragment. A total of 122 distance restraints derived from the difference spectra enabled the calculation of the structure of the bound peptide. The peptide forms a 10-residue loop, while the two segments flanking this loop interact extensively with each other and possibly form anti-parallel β-strands. The loop conformation could be observed due to the unusual large size (17 residues) of the antigenic determinant recognized by 0.5β.     

Conformational study of α1-acid glycoprotein

The secondary structure of the protein moiety of the α₁-acid glycoprotein (orosomucoid) was evaluated from its primary structure by using the Lim and Chou and Fasman predictions, and the corresponding dichroic spectrum was calculated. The experimental dichroic spectrum of the whole glycoprotein was compared with the summation of (i) the calculated dichroic spectrum due to the protein moiety and (ii) the experimental dichroic spectrum of the carbohydrate moiety. The results are in good agreement with the fact that the carbohydrate moities do not produce any pertubation of the protein conformation. In addition, we observed that four out of five glycan chains are linked to Asn residues which are situated either in a reverse β-turn or in regions where charged and polar residues are numerous, that is, on the outside of the protein.