Conformational study of O-glycosylated threonine containing peptide models

1H n.m.r. studies of Z-Thr-OMe, Z-Thr-Ala-OMe, Z-Ala-Thr-OMe and their glycosylated derivatives indicate the possibility of an intramolecular hydrogen bond between Thr N alpha H and the N-acetyl carbonyl of the carbohydrate moiety, 3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-alpha-D-galactopyranose (AcGalNAc). This is especially true in the case of Z-Thr(AcGalNAc)-Ala-OMe, suggesting that the strength of this hydrogen bond is dependent on the neighboring amino acids on the carbonyl terminal side of Thr. The existence of such a hydrogen bond implies a conformation in which the carbohydrate moiety is restricted to an orientation with its plane roughly perpendicular to the peptide backbone. In such an orientation, steric problems will be minimized in the case of clustered O-glycosidically linked Thr(Ser) residues as found in human erythrocyte glycophorin. A locked orientation of the carbohydrate moiety with respect to the peptide backbone may also play a conformational role in antifreeze glycoproteins.     

Conformational analysis of two somatostatin analogues by 1H 500 MHz n.m.r. spectroscopy

A series of nine closely related somatostatin analogues, containing the hexapeptide H-Cys2-Phe3-D-Trp4-Lys5-Thr6-Cys7-NH2 sequence have been synthesized by Bauer et al. The conformational properties of two of them, showing intermediate activities between those of SMS 201-995 and somatostatin, have been studied by high field n.m.r. spectroscopy in DMSO. Assignments were made using 2D-n.m.r. methods, in particular NOESY experiments and detection of long-range connectivities in aromatic residues. In all the compounds of this series, the biologically active ones as well as the inactive ones, the n.m.r. parameters are in favour of a predominant conformation with a type II' beta turn involving amino acids Phe3 to Thr6. A clearcut correlation exists between the predominant conformation at the cystine bridge side and the activity. The presence of the exocyclic amino acids Phe1 and Thr8 (ol) plays a major role in stabilization of the active conformation.     

Conformation and mobility of the RNA-binding N-terminal part of the intact coat protein of cowpea chlorotic mottle virus. A two-dimensional proton nuclear magnetic resonance study

Two-dimensional proton nuclear magnetic resonance (n.m.r.) experiments were performed on the coat protein of cowpea chlorotic mottle virus (molecular mass: 20.2 kDa) present as dimer (pH 7.5) or as capsid consisting of 180 protein monomers (pH 5.0). The spectra of both dimers and capsids showed resonances originating from the flexible N-terminal region of the protein. The complete resonance assignment of a synthetic pentacosapeptide representing this N terminus made it possible to interpret the spectra in detail. The capsid spectrum showed backbone amide proton resonances arising from the first eight residues having a flexible random coil conformation, and side-chain resonances arising from the first 25 N-terminal amino acids. The dimer spectrum showed also side-chain resonances of residues 26 to 33, which are flexible in the dimer but immobilized in the capsid. The n.m.r. experiments indicated that the conformation of the first 25 amino acids of the protein in dimers and capsids is comparable to the conformation of the synthetic peptide, which alternates among extended and helical conformations on the n.m.r. time-scale. It is suggested that the alpha-helical region, situated in the region between residues 10 and 20, binds to the RNA during assembly of the virus particle.     

Effects of glycosylation on the conformation and dynamics of O-linked glycoproteins: carbon-13 NMR studies of ovine submaxillary mucin

Carbon-13 NMR spectroscopic studies of native and sequentially deglycosylated ovine submaxillary mucin (OSM) have been performed to examine the effects of glycosylation on the conformation and dynamics of the peptide core of O-linked glycoproteins. OSM is a large nonglobular glycoprotein in which nearly one-third of the amino acid residues are Ser and Thr which are glycosylated by the alpha-Neu-NAc(2-6)alpha-GalNAc- disaccharide. The beta-carbon resonances of glycosylated Ser and Thr residues in intact and asialo mucin display considerable chemical shift heterogeneity which, upon the complete removal of carbohydrate, coalesces to single sharp resonances. This chemical shift heterogeneity is due to peptide sequence variability and is proposed to reflect the presence of sequence-dependent conformations of the peptide core. These different conformations are thought to be determined by steric interactions of the GalNAc residue with adjacent peptide residues. The absence of chemical shift heterogeneity in apo mucin is taken to indicate a loss in the peptide-carbohydrate steric interactions, consistent with a more relaxed random coiled structure. On the basis of the 13C relaxation behavior (T1 and NOE) the dynamics of the alpha-carbons appear to be unique to each amino acid type and glycosylation state, with alpha-carbon mobilities decreasing in the order Gly greater than Ala = Ser greater than Thr much greater than monoglycosylated Ser/Thr approximately greater than disaccharide linked Ser/Thr.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and conformation of cyclic hexapeptide cyclo(Pro-Sar-Sar)2

A cyclic hexapeptide, cyclo(Pro-Sar-Sar)₂, which consists of N-substituted amino acids only was synthesized, and its solution conformation was investigated by n.m.r. spectroscopy. Seven different C₂-symmetric conformations were detected, which were distinguishable from each other on the n.m.r. time scale. This is due to the cis/trans isomerization of N-substituted peptide bonds. Allowed C₂-symmetric conformations were computed on the basis of a hard-sphere model. Some conformations detected in n.m.r. spectra were not allowed in the calculation. This disagreement suggests that some asymmetric conformations with regard to the single bond rotation are averaged out due to a rapid rotation on the n.m.r. time scale. These points indicate that the molecule of cyclo (Pro-Sar-Sar)₂ is very flexible     

SMS 201-995, an octapeptide somatostatin analogue. Assignment of the 1H 500 MHZ n.m.r. spectra and conformational analysis of SMS 201-995 in dimethylsulfoxide

The possible conformations of SMS 201-995, an active analogue of somastostatin, have been studied in dimethylsulfoxide solution by 500 MHz proton n.m.r. spectroscopy. The assignments have been made by use of 2D-correlated methods to detect long-range coupling connectivities in aromatic residues and between the alpha protons of consecutive residues. NOESY experiments enabled us to correlate amide and alpha protons of neighbouring amino acid residues, which indicate a less flexible situation than in water. Measurements of temperature coefficients of the amide protons, of NH-C alpha H coupling constants and NOE effects are in favour of one predominant conformation with a beta turn, of type II', involving amino acids Phe3 to Thr6.     

Conformation analysis of D-xylose-containing O-glycopeptide sequences]

The preferred conformations of the sequences of O-glycopeptides containing a beta-D-xylosyl group linked to an L-seryl residue, found in the N-terminus of proteodermatan sulfate, show an almost extended peptide chain with the sugar unit in a specific position. The results of force-field calculations with the AMBER program have been confirmed, by n.m.r.-spectroscopic experiments, for a minimum conformation.     

Free energy landscapes of peptides by enhanced conformational sampling

The free energy landscapes of peptide conformations in water have been observed by the enhanced conformational sampling method, applying the selectively enhanced multicanonical molecular dynamics simulations. The conformations of the peptide dimers, -Gly-Gly-, -Gly-Ala-, -Gly-Ser-, -Ala-Gly-, -Asn-Gly-, -Pro-Gly-, -Pro-Ala-, and -Ala-Ala-, which were all blocked with N-terminal acetyl and C-terminal N-methyl groups, were individually sampled with the explicit TIP3P water molecules. From each simulation trajectory, we obtained the canonical ensemble at 300 K, from which the individual three-dimensional landscape was drawn by the potential of mean force using the three reaction coordinates: the backbone dihedral angle, psi, of the first amino acid, the backbone dihedral angle, phi, of the second amino acid, and the distance between the carbonyl oxygen of the N-terminal acetyl group and the C-terminal amide proton. The most stable state and several meta-stable states correspond to extended conformations and typical beta-turn conformations, and their free energy values were accounted for from the potentials of mean force at the states. In addition, the contributions from the intra-molecular energies of peptides and those from the hydration effects were analyzed. Consequently, the stable beta-turn conformations in the free energy landscape were consistent with the empirically preferred beta-turn types for each amino acid sequence. The thermodynamic values for the hydration effect were decomposed and they correlated well with the empirical values estimated from the solvent accessible surface area of each molecular conformation during the trajectories. The origin of the architecture of protein local fragments was analyzed from the viewpoint of the free energy and its decomposed factors.     

Synthetic TN glycopeptide related to human glycophorin AM. High-field proton and carbon-13 nuclear magnetic resonance study

Three 2-acetamido-2-deoxy-alpha-D-galactopyranoses attached to Ser2, Thr3 and Thr4 of the amino-terminal portion of glycophorin AM are responsible for the so-called TN blood group specificity. The corresponding glycopeptide H2N-Ser-Ser*-Thr*-Gly-OH obtained by a stepwise peptide coupling strategy was submitted to a detailed high-field nuclear magnetic resonance (n.m.r.) analysis. 13C-n.m.r. spectrum confirms the validity of previous assignments made on M sialo and asialoglycopeptides obtained by specific degradation of human glycophorin AM. In addition, the 400 MHz 1H-n.m.r. spectrum allowed most of the proton resonances to be assigned. A careful examination of the chemical shifts and coupling constants revealed some interesting features of the conformational properties of the GalNAc-Ser and GalNAc-Thr linkage as well as of the rotational isomerism of Thr and Ser side-chains. The data give conclusive evidence that high-field n.m.r. spectroscopy can be successfully used to gain structural and dynamic information on rather sophisticated glycopeptides.     

Synthesis and conformational analysis of Aib-containing peptide modelling for N-glycosylation site in N-glycoprotein

A tetrapetide containing an Aib residue, Boc-Asn-Aib-Thr-Aib-OMe, was synthesized as a peptide model for the N-glycosylation site in N-glycoproteins. Backbone conformation of the peptide and possible intramolecular interaction between the Asn and Thr side chains were elucidated by means of n.m.r. spectroscopy. Temperature dependence of NH proton chemical shift and NOE experiments showed that Boc-Asn-Aib-Thr-Aib-OMe has a tendency to form a β-turn structure with a hydrogen bond involving Thr and Aib⁴ NH groups. Incorporation of Aib residues in the peptide model promotes folding of the peptide backbone. With folded backbone conformation, carboxyamide protons of the Asn residue are not involved in hydrogen bond network, while the OH group of the Thr residue is a candidate for a hydrogen bond in DMSO-d₆ solution.     

Solution conformation of a peptide corresponding to the principal neutralizing determinant of HIV-1IIIB: a two-dimensional NMR study.

The 24 amino acid peptide RP135 corresponds in its amino acid sequence to the principal neutralizing determinant (PND) of the IIIB isolate of HIV-1. Although the sequence of the PND is highly variable, its central part, containing the sequence GPGR, is conserved in most HIV isolates. Using 2D NMR and CD spectroscopy, we have studied the conformation of RP135 and of two shorter versions: one (P547) that includes the GPGR sequence with the N-terminal part of the peptide and the other (P344) that includes GPGR and the C-terminal segment of RP135. In water, the C-terminal part of RP135 was found to exist in several transient turnlike conformations ("nascent helix"). A helical conformation was found to be stabilized by the addition of TFE. A transient turn was observed also in the GPGR sequence, both in water and in aqueous TFE solutions. While no nascent helix conformations could be observed in the N-terminal part of RP135 in water, a helical conformation was partially stabilized by the addition of TFE. The conformations of the two shorter versions of the peptide were similar to those of the corresponding parts of RP135, except that the transient turn in GPGR could not be detected in P547 dissolved in water. The turn in GPGR was previously predicted by Larosa et al. (1990) and was observed by Chandrasekhar et al. (1991) in the PND peptide of HIV-1MN (RP142), which shares only 56% identity with RP135. However, nascent helix conformations were not observed in aqueous solutions of RP142.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural modifications in the membrane-bound regions of the gastric H+/K+-ATPase upon ligand binding.

Extensive trypsin proteolysis was used to examine the accessibility of membrane bound segments of the gastric H+/K+-ATPase under different experimental conditions known to induce either the E1 or the E2 conformation. Membrane-anchored peptides were isolated after trypsinolysis and identified by sequencing. We show that several membrane bound segments are involved in the conformational change. In the N-terminal region, a M1-M2 peptide (12 kDa) was found to be associated with the membrane fraction after digestion in the presence of K+ or in the presence of vanadate (12 kDa and 15 kDa). In the M3 and M4 region, no difference was observed for the peptide obtained in E1 or E2-K conformations, but the peptide generated in the presence of vanadate begins 12 amino-acid residues earlier in the sequence. Cytoplasmic loop region: we show here that a peptide beginning at Asp574 and predicted to end at Arg693 is associated with the membrane for a vanadate-induced conformation. In the M5-M6 region, the membrane-anchored peptide obtained on E1 is 39 amino acids shorter than the E2 peptide. In the M7-M8 region, the same peptide encompassing the M7 and M8 transmembrane segments was produced for E1 and E2 conformations.     

Incompatible blood-group A determinants in tumoral mucins. Isolation of oligosaccharides having a 2-acetamido-2-deoxy-alpha-D-galactopyranosyl group at the non-reducing end

Two glycopeptide fractions were obtained from pseudomyxomatous mucins secreted by an ovarian cystadenocarcinoma from a female having blood-group B, and by an appendix tumor from a male having blood-group O. The carbohydrate and amino acid content of these fractions suggests the presence of numerous carbohydrate side-chains linked through O-glycosyl bonds to a peptide core rich in threonine and proline. The two glycopeptide fractions exhibit compatible B- and H-blood-group activities. They are reactive towards Dolichos biflorus lectin and human anti-A agglutinins, and so exhibit an incompatible A activity. Alkali-borohydride degradation of Pronase-digested glycopeptides gave dialyzable oligosaccharides that were purified and shown to possess 2-acetamido-2-deoxygalactitol at the terminal reducing-end. 2-Acetamido-2-deoxyglucose, galactose, fucose, and neuraminic acid were absent, or present, in variable proportions. Four oligosaccharides containing 2-acetamido-2-deoxy-D-galactose residues were reactive towards Dolichos biflorus lectin and human anti-A agglutinins, indicating the presence, at the nonreducing end, of a 2-acetamido-2-deoxy-alpha-D-galactopyranosyl group, responsible for blood-group A activity.     

Synthesis and conformational analysis of aib-containing peptide modelling for N-glycosylation site in N-glycoprotein

A tetrapetide containing an Aib residue, Boc-Asn-Aib-Thr-Aib-OMe, was synthesized as a peptide model for the N-glycosylation site in N-glycoproteins. Backbone conformation of the peptide and possible intramolecular interaction between the Asn and Thr side chains were elucidated by means of n.m.r. spectroscopy. Temperature dependence of NH proton chemical shift and NOE experiments showed that Boc-Asn-Aib-Thr-Aib-OMe has a tendency to form a β-turn structure with a hydrogen bond involving Thr and Aib⁴ NH groups. Incorporation of Aib residues in the peptide model promotes folding of the peptide backbone. With folded backbone conformation, carboxyamide protons of the Asn residue are not involved in hydrogen bond network, while the OH group of the Thr residue is a candidate for a hydrogen bond in DMSO-d₆ solution.     

Amino acid sequence of the signal peptide of mitochondrial nicotinamide nucleotide transhydrogenase as determined from the sequence of its messenger RNA

The amino acid sequence of the bovine mitochondrial nicotinamide nucleotide transhydrogenase was recently deduced from isolated cDNAs and reported [Yamaguchi, M., Hatefi, Y., Trach, K., and Hoch, J.A. (1988) J. Biol. Chem. 263, 2761-2767]. The cDNAs lacked the N-terminal coding region, however, and the 8 N-terminal residues were determined by protein sequencing. In the present study, the nucleotide sequence of the 5' upstream region was determined by dideoxynucleotide sequencing of the transhydrogenase messenger RNA, and amino acid sequences of the N-terminal region and the signal peptide of the enzyme were deduced from the nucleotide sequence. The N-terminal sequence of the enzyme as deduced from the mRNA sequence is the same as that determined by protein sequencing, with one difference. Protein sequencing showed Ser as the N-terminal residue. The mRNA sequence indicated that Ser is the second N-terminal residue, and the first is Cys. That preparations of the enzyme are mixtures of two polypeptides, one polypeptide being one residue shorter at the N terminus than the other, has been pointed out in the above reference. The signal peptide consists of 43 residues, is rich in basic (4 Lys, 2 Arg) and hydroxylated (4 Thr, 3 Ser) amino acids, and lacks acidic residues.     

C n.m.r. study of the pH behaviour of N-methylated peptides related to the N-terminus of glycophorins

¹³C nuclear magnetic resonance spectroscopy (¹³C n.m.r.) was used to determine the pH titration parameters for the N-terminal N,N-[¹³C]dimethylamino and N,N-[¹³C]monomethylamino groups of glycophorins A^M and A^N, and some 28 related glycoproteins, glycopeptides and peptides. The results show that glycosylation of the Ser and Thr residues at positions 2, 3 and 4 of the glycophorins have a pronounced effect on the titration parameters. Substitution of amino acids 4 and 5 in the glycophorin sequence appears to minimally affect our titration parameters. Internal hydrogen-bonding involving the N-terminal Ser residue may explain some of the unusual pH titration results observed for glycophorin A^M.     

Database analysis of O-glycosylation sites in proteins

Statistical analysis was carried out to study the sequential aspects of amino acids around the O-glycosylated Ser/Thr. 992 sequences containing O-glycosylated Ser/Thr were selected from the O-GLYCBASE database of O-glycosylated proteins. The frequency of occurrence of amino acid residues around the glycosylated Ser/Thr revealed that there is an increased number of proline residues around the O-glycosylation sites in comparison with the nonglycosylated serine and threonine residues. The deviation parameter calculated as a measure of preferential and nonpreferential occurrence of amino acid residues around the glycosylation site shows that Pro has the maximum preference around the O-glycosylation site. Pro at +3 and/or -1 positions strongly favors glycosylation irrespective of single and multiple glycosylation sites. In addition, serine and threonine are preferred around the multiple glycosylation sites due to the effect of clusters of closely spaced glycosylated Ser/Thr. The preference of amino acids around the sites of mucin-type glycosylation is found likely to be similar to that of the O-glycosylation sites when taken together, but the acidic amino acids are more preferred around Ser/Thr in mucin-type glycosylation when compared totally. Aromatic amino acids hinder O-glycosylation in contrast to N-glycosylation. Cysteine and amino acids with bulky side chains inhibit O-glycosylation. The preference of certain potential sequence motifs of glycosylation has been discussed.     

An efficient access to protected disialylated glycohexaosyl threonine present on the leukosialin of activated T-lymphocytes

The total synthesis of the threonine-linked core 2 class disialylated hexasaccharide in a completely protected form was accomplished for the first time. The L-threonine conjugate, N-(9-fluorenylmethoxycarbonyl)-O-[(5-acetamido-4,7,8,9-tetra-O-ben zyl-3,5-dideoxy-D-glycero-alpha-D-galacto-2-nonulopyranosylonic acid)-(2-->3)-(2,6-di-O-benzyl-beta-D-galactopyranosyl)-(1-->4)-2-acetam ido-2-deoxy-3,6-di-O-benzyl-beta-D-glucopyranosyl-(1-->6)-[(5-acetamido- 4,7,8,9-tetra-O-benzyl-3,5-dideoxy-D-glycero-alpha-D-galacto-2-nonulo pyranosylonic acid)-(2-->3)-2,6-di-O-benzyl-beta-D-galactopyranosyl-(1-->3)]-2-acetami do-2-deoxy-alpha-D-galactopyranosyl-(1d-->4c:1f-->4e)-dilactone ]-L-threonine allyl ester was synthesized via stereocontrolled glycosylations employing readily accessible monosaccharidic blocks; t-butyl-diphenylsilyl-2-azido-2-deoxy-3,6-di-O-benzyl-beta-D-gluco pyranose, N-(9-fluorenylmethoxycarbonyl)-O-(2-azido-6-O-t-butyldimethylsilyl -2-deoxy-alpha-D-galactopyranosyl)-L-threonine allyl ester, 8, 9 and N-(9-fluorenylmethoxycarbonyl)-O-(2-azido-4,6-O-benzylidene-3-O-ch loroacetyl-2-deoxy-alpha-D-galactopyranosyl)-L-threonine allyl ester. For the introduction of the amino acid, the azide group was used to temporarily mask the amino group of GalNAc so as to obtain an alpha-glycosidic linkage without participation from the C-2 substituent. The threonine was attached to the sugar unit at the monosaccharide stage to avoid loss of oligosaccharide at a later stage. The Fmoc and allyl ester protected amino acid at the reducing end facilitates efficient glycopeptide synthesis on solid-phase support.     

Conformations of dehydrophenylalanine containing peptides. Nuclear Overhauser effect study of two acyclic tetrapeptides

Two isomeric, acyclic tetrapeptides containing a Z-dehydrophenylalanine residue (delta Z-Phe) at position 2 or 3, Boc-Leu-Ala-delta Z-Phe-Leu-OMe (1) and Boc-Leu-delta Z-Phe-Ala-Leu-OMe (2), have been synthesized and their solution conformations investigated by 270 MHz 1H n.m.r. spectroscopy. In peptide 1 the Leu(4) NH group appears to be partially shielded from solvent, while in peptide 2 both Ala(3) and Leu(4) NH groups show limited solvent accessibility. Extensive difference nuclear Overhauser effect (n.O.e.) studies establish the occurrence of several diagnostic inter-residue n.O.e.s (Ci alpha H----Ni+1H and NiH----Ni+1H) between backbone protons. The simultaneous observation of "mutually exclusive" n.O.e.s suggests the presence of multiple solution conformations for both peptides. In peptide 1 the n.O.e. data are consistent with a dynamic equilibrium between an -Ala-delta Z-Phe- Type II beta-turn structure and a second species with delta Z-Phe adopting a partially extended conformation with psi values of +/- 100 degrees to +/- 150 degrees. In peptide 2 the results are compatible with an equilibrium between a highly folded consecutive beta-turn structure for the -Leu-delta Z-Phe-Ala- segment and an almost completely extended conformation.