Novel properties of peptides derived from the sequence coded by exon 26A of human elastin.

The exon 26A is a rarely expressed human elastin exon that codes for a hydrophilic and charged amino acid sequence. The functional role of elastin containing this additional sequence is unknown. The present investigation was aimed to determine the effect of synthetic peptides derived from this exon on the vascular tone of rat thoracic aorta. On phenilephrine-preconstricted rat thoracic aortic rings the peptides LSPELREGD and REGD cause dose-dependent relaxation in the concentration range from 10(-9) to 10(-5) M. omega-nitro-L-arginine methyl ester, a known inhibitor of the NO synthase, highly inhibits, although to a different extent, the relaxation induced by these peptides. Removal of endothelium and blocking of ATP-sensitive potassium channels by glibenclamide significantly inhibited the vasorelaxant activity of LSPELREGD but not that of REGD, suggesting a different mechanism of action and possibly a different receptor.     

Structural characterization of human elastin derived peptides containing the GXXP sequence

The degradation of elastin, the insoluble biopolymer of tropoelastin, can lead to the production of small peptides. These elastin-derived peptides (EDPs) are playing a key role in cellular behavior within the extracellular matrix, showing a great variety of biological effects such as chemotaxis, stimulation of cell proliferation, ion flux modifications, vasorelaxation, and inflammatory enzymes secretion. It has also been demonstrated recently that EDPs containing the GXXPG motif could induce pro-MMP1 and pro-MMP3 upregulation. Elastolysis could then cause collagen degradation and play an important role in the aging process. Many experimental studies have been devoted to EDPs, but their structure/activity relationships are not well elucidated yet. However, the assumption that their active conformation is a type VIII beta-turn on GXXP was highly suggested on the basis of predictive statistical calculations. Investigation of the EDPs three-dimensional (3D) structure would provide useful information for drug-design strategies to propose specific inhibitors. The work presented here reports theoretical results obtained from molecular dynamics simulations performed over 128 human EDPs containing the GXXP motif. We show that all the peptides, for which the central residues are not glycines, adopt a canonical (or very close to) type VIII beta-turn structure on the GXXP sequence. Amino acids surrounding this motif are also important for the structural behavior. Any residue located before the GXXP motif (XGXXP) increases the beta-turn stabilization, whereas the residue located after GXXP (GXXPX) has no significant structural effect. Moreover, we show their biological activity can be correlated with their ability to exhibit a type VIII beta-turn conformation.     

A turning point in the knowledge of the structure-function-activity relations of elastin]

In this review are presented the last new results of our research group dealing with the molecular structures (atomic level) of tropoelastin, elastin and elastin derived peptides studied by using essentially methods of bioinformatics (theoretical predictions and molecular modelling) linked to experimental circular dichroism spectroscopic studies. We already had characterized both the local secondary structure and some parts of the tertiary structure of the tropoelastin and elastin molecules (human, bovine...), by using either theoretical predictions (local secondary structure, linear epitopes...) and/or experimental data (optical spectroscopic methods: Raman scattering, infrared absorption, circular dichroism). Except the cross-linking regions which are in helical conformations, the whole tropoelastin structure displays a lot of beta-reverse turns which usually belong to irregular structures in proteins. These turns play a key role in other regularly structures orientation (alpha-helix, beta-strand), thus they are very important in the native protein 3D architecture. It is particularly true for human tropoelastin, because its sequence is rich in glycines and prolines, and these residues are frequently met in beta-turns (a beta-turn is made of four consecutive residues which are stabilized by an hydrogen bond). Several types of beta-turns can be defined with the dihedral angles values phi and psi of the two central residues. Thus, by using a very recent updated set of propensities for the amino acid residues to belong to given types of reverse beta-turns (extracted from a reference set of known 3-D structures of globular proteins), we have determined, (by using our home made software COUDES), for all possible tetrapeptides of the human tropoelastin sequence, the distribution and the characterization of the possible type of turns. Thus, it is shown that the locations and/or the types of these reverse beta-turns reveal a regularity and are not all random. This confirms our hypothesis that intra-molecular elasticity of tropoelastin could be explained by the possibility of transitions between conformations involving short beta-strands and beta-turns. This result is of great interest in the construction (by using molecular biology) of elastic biomaterials derived from the elastin sequence (particularly, the elastin derived peptides corresponding to the sequence exon 21--(exon 24--exon 24...). Our study permit also to predict the conformations of specific elastin derived peptides which could have interesting biological activity. Peptides resulting from the degradation of elastin, the insoluble polymer of tropoelastin and responsible for the elasticity of vertebrate tissues, can induce biological effects and notably the regulation of matrix metalloproteinases (MMP-s) activity. Recently, it was proposed that some elastin derived hexapeptides resulting from circular permutations of VGVAPG (a three fold repetition sequence in exon 24 of human tropoelastin) possess MMP-1 production and activation regulation properties. This effect depends on the presence of the tropoelastin specific membraneous receptor 67 KDa EBP (Elastin Binding Protein). Our results obtained by using both circular dichroism spectroscopy and linear predictions confirmed the hypothesis of a structure dependent mechanism with a possibly occurring type VIII beta-turn on the first four residues of the GXXPG sequence consensus which is only present among all active peptides. Thus, we have performed extensive molecular dynamics studies, in both implicit and explicit solvent, on these active and inactive elastin derived hexapeptides. Using our own analysis method of pattern recognition of the types of the beta-reverse-turns followed during the molecular dynamics trajectory, we found that active and inactive peptides effectively form two well distinct conformational groups in which active peptides preferentially adopt conformation close to type VIII GXXP (beta-reverse-turn. The structural role of the C terminal G residue could also be explained. Additional molecular simulations on (VGVAPG)2 and (VGVAPG)3 show the formation of two or three GXXP tetrapeptides adopting a structure close to type VIII beta-reverse-turn, suggesting a local conformational preference for this motif. This observation of a specific structural single and/or repeated motif is in agreement with the circular dichroism spectra of the involved (VGVAPG)1, (VGVAPG)2 and (VGVAPG)3 peptides and then it can be proposed that their biological activities have to be linear. The final aim of this type of work is to understand more about the sequence/structure/function/activity relationships of those structured peptides in order to propose specific sequences (corresponding to specific structures) for best biological activity results.     

Structure characterization of the central repetitive domain of high molecular weight gluten proteins. I. Model studies using cyclic and linear peptides.

The high molecular weight (HMW) proteins from wheat contain a repetitive domain that forms 60-80% of their sequence. The consensus peptides PGQGQQ and GYYPTSPQQ form more than 90% of the domain; both are predicted to adopt beta-turn structure. This paper describes the structural characterization of these consensus peptides and forms the basis for the structural characterization of the repetitive HMW domain, described in the companion paper. The cyclic peptides cyclo-[PGQGQQPGQGQQ] (peptide 1), cyclo-[GYYPTSPQQGA] (peptide 2), and cyclo-[PGQGQQGYYPTSPQQ] (peptide 3) were prepared using a novel synthesis route. In addition, the linear peptides (PGQGQQ)n (n = 1, 3, 5) were prepared. CD, FTIR, and NMR data demonstrated a type II beta-turn structure at QPGQ in the cyclic peptide 1 that was also observed in the linear peptides 9PGQGQQ)n. A type I beta-turn was observed at YPTS and SPQQ in peptides 2 and 3, with additional beta-turns of either type I or II at GAGY (peptide 2) and QQGY (peptide 3). The proline in YPTS showed considerable cis/trans isomerization, with up to 50% of the population in the cis-conformation; the other prolines were more than 90% in the trans conformation. The conversion from trans to cis destroys the type I beta-turn at YPTS, but leads to an increase in turn character at SPQQ and GAGY (peptide 2) or QQGY (peptide 3).     

Structure, biological activity and membrane partitioning of analogs of the isoprenylated a-factor mating peptide of Saccharomyces cerevisiae.

Previous biochemical investigations on the Saccharomyces cerevisiae a-factor indicated that this lipopeptide pheromone [YIIKGVFWDPAC(farnesyl)OMe] might adopt a type II beta-turn at positions 4 and 5 of the peptide sequence. To test this hypothesis, we synthesized five analogs of a-factor, in which residues at positions 4 and 5 were replaced with: L-Pro4(I); D-Pro4(II); L-Pro4-D-Ala5(III); D-Pro4-L-Ala5(IV); or Nle4(V). Analogs were purified to > 99% homogeneity as evidenced by HPLC and TLC and were characterized by mass spectrometry and amino acid analysis. Using a growth arrest assay the conformationally restricted a-factor analogs I and III were found to be almost 50-fold more active than the diastereometric homologs II and IV and were equally active to wild-type a-factor. Replacement of Lys4 with the isosteric Nle4 almost abolished the activity of the pheromone. Thus, the incorporation of residues that promote a type II beta-turn compensated for the loss of the favorable contribution of the Lys4 side chain to pheromone activity. CD spectra on these peptides suggested that they were essentially disordered in both TFE/H2O and in the presence of DMPC vesicles. There was no correlation between CD peak shape and biological activity. Using fluorescence spectroscopy we measured the interaction of lipid vesicles with these position 4 and 5 analogs as well as with three a-factor analogs with a modified farnesyl group. The results indicated that modifications of both the peptide sequence and the lipid moiety affect partitioning into lipid, and that no correlation existed between the propensity of a pheromone to partition into the lipid and its biological activity.     

A comparative 1H-n.m.r. study of MurNAc-L-Ala-D-iGln (MDP) and its analogue murabutide: evidence for a structure involving two successive beta-turns in MDP

The active principle, MurNAc-L-Ala-D-iGln (MDP), of complete Freund's adjuvant and its analogue, MurNAc-L-Ala-D-Gln-OnBu (murabutide), which express immunomodulatory as well as other biological properties, have been studied by 2D-1H-n.m.r. spectroscopy at 500 MHz. The results suggest the presence in MDP of two successive turns involving the MurNAc-L-Ala and L-Ala-D-iGln moieties, respectively, whereas only the former turn persists in murabutide. This turn mimics the type II beta-turn found in L-D depsipeptides, whereas the other is a typical type II beta-turn for L-D peptides.     

Immunosuppressive analogues of hexapeptide Tyr-Val-Pro-Leu-Phe-Pro, an immune system stimulant

It was found that substitution of Val2 and/or Leu4 residue in a hexapeptide Tyr-Val-Pro-Leu-Phe-Pro (I) transforms this peptide immunostimulant into analogues possessing the immunosuppressor activity--Tyr-Gly-Pro-Leu-Phe-Pro (II), Tyr-Val-Pro-Gly-Phe-Pro (III), and Tyr-Gly-Pro-Gly-Phe-Pro (IV). Biological effects of peptides I-IV were studied using PFC (plaque forming cell) test, GvH (graft vs host) reaction in mice, and ARFC (autologous rosette forming cell) test. The strongest immunosuppressor activity was observed for III--in this case the immunosuppressor effect was observed even in PFC test in vitro in which II and IV showed no such activity. These results suggest that simultaneous presence of both Val2 and Leu4 residues is necessary for the generation of immunostimulation. The CD study of I-IV in methanol solution suggests that the conformational preferences of II-IV change towards stabilization of the beta-turn structure, whereas in the case of I the gamma-turn on Leu4 and cis' orientation of the Pro3 carbonyl (distorted beta-turn of type I) was found as the preferred conformation. Competition in biological effects observed for I and III in PFC in vitro test suggests that these analogues may interact with the same cellular receptor. Drastic changes in the activity which accompany changes in the sequence are discussed in the terms of our stereochemical hypothesis.     

The antitumor properties of the alpha3(IV)-(185-203) peptide from the NC1 domain of type IV collagen (tumstatin) are conformation-dependent

Tumor progression may be controlled by various fragments derived from noncollagenous 1 (NC1) C-terminal domains of type IV collagen. We demonstrated previously that a peptide sequence from the NC1 domain of the alpha3(IV) collagen chain inhibits the in vitro expression of matrix metalloproteinases in human melanoma cells through RGD-independent binding to alpha(v)beta(3) integrin. In the present paper, we demonstrate that in a mouse melanoma model, the NC1 alpha3(IV)-(185-203) peptide inhibits in vivo tumor growth in a conformation-dependent manner. The decrease of tumor growth is the result of an inhibition of cell proliferation and a decrease of cell invasive properties by down-regulation of proteolytic cascades, mainly matrix metalloproteinases and the plasminogen activation system. A shorter peptide comprising the seven N-terminal residues 185-191 (CNYYSNS) shares the same inhibitory profile. The three-dimensional structures of the CNYYSNS and NC1 alpha3(IV)-(185-203) peptides show a beta-turn at the YSNS (188-191) sequence level, which is crucial for biological activity. As well, the homologous MNYYSNS heptapeptide keeps the beta-turn and the inhibitory activity. In contrast, the DNYYSNS heptapeptide, which does not form the beta-turn at the YSNS level, is devoid of inhibitory activity. Structural studies indicate a strong structure-function relationship of the peptides and point to the YSNS turn as necessary for biological activity. These peptides could act as potent and specific antitumor antagonists of alpha(v)beta(3) integrin in melanoma progression.     

Structure-function analysis of tritrypticin, an antibacterial peptide of innate immune origin.

The structural requirements for the antibacterial activity of a pseudosymmetric 13-residue peptide, tritrypticin, were analyzed by combining pattern recognition in protein structures, the structure-activity knowledge-base, and circular dichroism. The structure-activity analysis, based on various deletion analogs, led to the identification of two minimal functional peptides, which by themselves exhibit adequate antibacterial activity against Escherichia coli and Salmonella typhimurium. The common features between these two peptides are that they both share an aromatic-proline-aromatic (ArProAr) sequence motif, and their sequences are retro with respect to one another. The pattern searches in protein structure data base using the ArProAr motif led to the identification of two distinct conformational clusters, namely polyproline type II and beta-turn, which correspond to the observed solution structures of the two minimal functional analogs. The role of different residues in structure and function of tritrypticin was delineated by analyzing antibacterial activity and circular dichroism spectra of various designed analogs. Three main results arise from this study. First, the ArProAr sequence motif in proteins has definitive conformational features associated with it. Second, the two minimal bioactive domains of tritrypticin have entirely different structures while having equivalent activities. Third, tritrypticin has a beta-turn conformation in solution, but the functionally relevant conformation of this gene-encoded peptide antibiotic may be an extended polyproline type II.     

Probing the conformational and dynamical effects of O-glycosylation within the immunodominant region of a MUC1 peptide tumor antigen.

MUC1 mucin is a large transmembrane glycoprotein, the extracellular domain of which is formed by a repeating 20 amino acid sequence, GVTSAPDTRPAPGSTAPPAH. In normal breast epithelial cells, the extracellular domain is densely covered with highly branched complex carbohydrate structures. However, in neoplastic breast tissue, the extracellular domain is under-glycosylated, resulting in the exposure of a highly immunogenic core peptide epitope (PDTRP in bold above), as well as in the exposure of normally cryptic core Tn (GalNAc), STn (sialyl alpha2-6 GalNAc) and TF (Gal beta1-3 GalNAc) carbohydrates. Here, we report the results of 1H NMR structural studies, natural abundance 13C NMR relaxation measurements and distance-restrained MD simulations designed to probe the structural and dynamical effects of Tn-glycosylation within the PDTRP core peptide epitope. Two synthetic peptides were studied: a nine-residue MUC1 peptide of the sequence, Thr1-Ser2-Ala3-Pro4-Asp5-Thr6-Arg7-Pro8-Ala9, and a Tn-glycosylated version of this peptide, Thr1-Ser2-Ala3-Pro4-Asp5-Thr6(alphaGalNAc)-Arg7-Pro8-Ala9. The results of these studies show that a type I beta-turn conformation is adopted by residues PDTR within the PDTRP region of the unglycosylated MUC1 sequence. The existence of a similar beta-turn within the PDTRP core peptide epitope of the under-glycosylated cancer-associated MUC1 mucin protein might explain the immunodominance of this region in vivo, as the presence of defined secondary structure within peptide epitope regions has been correlated with increased immunogenicity in other systems. Our results have also shown that Tn glycosylation at the central threonine within the PDTRP core epitope region shifts the conformational equilibrium away from the type I beta-turn conformation and toward a more rigid and extended state. The significance of these results are discussed in relation to the possible roles that peptide epitope secondary structure and glycosylation state may play in MUC1 tumor immunogenicity.     

Conformation-activity relationship of peptide T and new pseudocyclic hexapeptide analogs.

Peptide T (ASTTTNYT), a segment corresponding to residues 185-192 of gp120, the coat protein of HIV, has several important biological properties in vitro that have stimulated the search for simpler and possibly more active analogs. We have previously shown that pseudocyclic hexapeptide analogs containing the central residues of peptide T retain considerable chemotactic activity. We have now extended the design of this type of analogs to peptides containing different aromatic residues and/or Ser in lieu of Thr. The complex conformation-activity relationship of these analogs called for a reexamination of the basic conformational tendencies of peptide T itself. Here, we present an exhaustive NMR conformational study of peptide T in different media. Peptide T assumes a gamma-turn in aqueous mixtures of ethylene glycol, a type-IV beta-turn conformation in aqueous mixtures of DMF, and a type-II beta-turn conformation in aqueous mixtures of DMSO. The preferred conformations for the analogs were derived from modeling, starting from the preferred conformations of peptide T. The best models derived from the gamma-turn conformation of peptide T are those of peptides XII (DSNYSR), XIII (ETNYTK) and XVI (ESNYSR). The best models derived from the type-IV beta-turn conformation of peptide T are those of peptides XIV (KTTNYE) and XV (DSSNYR). No low-energy models could be derived starting from the type-II beta-turn conformation of peptide T. The analogs with the most favored conformations are also the most active in the chemotactic test.     

Identification of conserved residue patterns in small beta-barrel proteins

Our abilities to predict three-dimensional conformation of a polypeptide, given its amino acid sequence, remain limited despite advances in structure analysis. Analysis of structures and sequences of protein families with similar secondary structural elements, but varying topologies, might help in addressing this problem. We have studied the small beta-barrel class of proteins characterized by four strands (n = 4) and a shear number of 8 (S = 8) to understand the principles of barrel formation. Multiple alignments of the various protein sequences were generated for the analysis. Positional entropy, as a measure of residue conservation, indicated conservation of non-polar residues at the core positions. The presence of a type II beta-turn among the various barrel proteins considered was another strikingly invariant feature. A conserved glycyl-aspartyl dipeptide at the beta-turn appeared to be important in guiding the protein sequence into the barrel fold. Molecular dynamics simulations of the type II beta-turn peptide suggested that aspartate is a key residue in the folding of the protein sequence into the barrel. Our study suggests that the conserved type II beta-turn and the non-polar residues in the barrel core are crucial for the folding of the protein's primary sequence into the beta-barrel conformation.     

Peptide T revisited: conformational mimicry of epitopes of anti-HIV proteins.

Peptide T (ASTTTNYT), a fragment corresponding to residues 185-192 of gp120, the coat protein of HIV, is endowed with several biological properties in vitro, notably inhibition of the binding of both isolated gp120 and HIV-1 to the CD4 receptor, and chemotactic activity. Based on previous nuclear magnetic resonance (NMR) studies performed in our laboratory, which were consistent with a regular conformation of the C-terminal pentapeptide, and SAR studies showing that the C-terminal pentapeptide retains most of the biological properties, we designed eight hexapeptides containing in the central part either the TNYT or the TTNY sequence, and charged residues (D/E/R) at the two ends. Conformational analysis based on NMR and torsion angle dynamics showed that all peptides assume folded conformations. albeit with different geometries and stabilities. In particular, peptides carrying an acidic residue at the N-terminus and a basic residue at the C-terminus are characterized by stable helical structures and retain full chemotactic activity. The solution conformation of peptide ETNYTR displays strong structural similarity to the region 19-26 of both bovine pancreatic and bovine seminal ribonuclease, which are endowed with anti-HIV activity. Moreover, the frequent occurrence, in many viral proteins, of TNYT and TTNY, the two core sequences employed in the design of the hexapeptides studied in the present work, hints that the sequence of the C-terminal pentapeptide TTNYT is probably representative of a widespread viral recognition motif.     

A computergraphic determination of the chemotactic peptide preferred conformation

Replacement of leucine in the chemotactic peptide For-Met-Leu-Phe by the sterically constrained amino acids alpha-aminoisobutyric acid and aminocyclohexanecarboxylic acid affords compounds of equal or greater activity than the parent. NMR studies indicate that the parent compound is present as a beta-sheet in solution, whereas the analogues prefer a beta-turn. Application of molecular modelling would indicate that the beta-turn conformer is energetically preferable and thus suggests that it is the orientation adopted by the peptides.     

Folding of immunogenic peptide fragments of proteins in water solution. I. Sequence requirements for the formation of a reverse turn

A systematic examination by 1H nuclear magnetic resonance of the population of beta-turn-containing conformers in several series of short linear peptides in water solution has demonstrated a dependence on amino acid sequence which has important implications for initiation of protein folding. The peptides consist of a number of variants of the sequence Tyr-Pro-Tyr-Asp, the trans isomer of which was previously shown to contain a reverse turn in water. Two-dimensional rotating-frame nuclear Overhauser effect spectroscopy provides unequivocal evidence that substantial populations of reverse turn conformations occur in water solutions of certain of these peptides. In the unfolded state, the peptides adopt predominantly extended chain (beta) conformations in water. It appears probable from the nuclear Overhauser effect connectivities observed that the reverse turns in the trans isomers are predominantly type II. The low temperature coefficient of the amide proton resonance of the residue at position 4 of the turn suggests the presence of an intramolecular hydrogen bond. The presence of the beta-turn conformation has been confirmed for certain peptides by circular dichroism measurements. Substitutions at positions 3 and 4 in the sequence Tyr-Pro-Tyr-Asp-Val can enhance or abolish the beta-turn population in the trans peptide isomers. The residue at position 3 of the turn is the primary determinant of its stability. A small amount of additional stabilization appears to result from an electrostatic interaction between the side-chain of residue 4 and the unblocked amino terminus. For peptides of the series Tyr-Pro-X-Asp-Val, where X represents all L-amino acid except Trp and Pro, the temperature coefficient of the Asp4 amide proton resonance provides a measure of the beta-turn population. The beta-turn populations in water solution measured in this way correlate with the beta-turn probabilities determined from protein crystal structures. This indicates that it is frequently the local amino acid sequence, rather than medium- to long-range interactions in the folded protein, that determines the beta-turn conformation in the folded state. Such sequences are excellent candidates for protein folding initiation sites. A high population of structured forms appears to be present in the cis isomer of certain of the peptides, as shown by a considerable increase in the proportion of the cis isomer and by measurement of nuclear Overhauser effects and 3JN alpha coupling constants.(ABSTRACT TRUNCATED AT 400 WORDS)     

Neutrophil chemotactic activity of N-terminal peptides from the calpain small subunit

On the basis of previous findings that N-acetyl nonapeptide from the human calpain I large subunit has chemotactic activity for neutrophils, a series of peptides with the N-terminal sequence of the calpain small subunit were synthesized and their chemotactic activity was examined. Potent activity was found in N-acetyl tetra, hepta, octa, nona and a larger peptide of 13 residues, although N-acetyl tripeptide showed only weak activity and N-acetyl penta and hexa peptides showed almost no activity. Since the small subunit is identical in calpains I and II, the results indicate that both calpains could be precursor proteins of chemotactic factors for neutrophils.     

Determination of structural elements related to the biological activities of a potent decapeptide agonist of human C5a anaphylatoxin.

The structural features related to the biologic activities of a potent, response-selective decapeptide agonist of human C5a, YSFKPMPLaR (C5a65-74, Y65, F67, P69, P71, D-Ala73), were identified by NMR analysis in H2O, DMSO and TFE. This investigation showed that the KPM residues in H2O and the SFKPM residues in DMSO exhibited an extended backbone conformation, whereas a twisted conformation was found in this region in TFE. In H2O, the C-terminal region (PLaR) adopted a distorted type II beta-turn or a type II/V beta-turn. In the type IIN beta-turn, Leu72 exhibited a conformation typical of a type II beta-turn, whereas D-Ala73 exhibited a conformation characteristic of a type V beta-turn. Furthermore, a gamma-turn involving residues LaR overlapped with the type II/V beta-turn. In DMSO, the C-terminal region had the analogous turn-like motif (type II/V beta-turn overlapping with gamma-turn) found in H2O. In TFE, no beta-turn motifs were formed by the PLaR residues. These turn-like motifs in the C-terminal region of the peptide in both H2O and DMSO were in agreement with the biologically important conformations predicted earlier by a structure-function analysis of a related panel of decapeptide analogs. The motifs determined by the NMR analysis of YSFKPMPLaR in H2O and DMSO may represent structural elements important for C5a agonist activity and thus can be used to design the next generation of C5a agonist, partial agonist and antagonist analogs.     

Effect of D-amino acid substitution in a mucin 2 epitope on mucin-specific monoclonal antibody recognition

We have studied the influence of D-amino acid substitution in the flanking region on the antibody recognition of the 19TGTQ22 epitope core in the tandem repeat of mucin 2 (MUC2) glycoprotein. Analogue peptides corresponding to the optimal epitope sequence (16PTPTGTQ22) have been prepared by the replacement of single or multiple L-amino acid residues at the N-terminal part of the molecule. According to previous studies, this portion of the all-L 16PTPTGTQ22 peptide possesses a beta-turn secondary structure important for efficient monoclonal antibody interaction. The binding properties of sequentially modified peptides (pTPTGTQ, ptPTGTQ, ptpTGTQ, and ptptGTQ) have been analyzed by a MUC2 glycoprotein specific monoclonal antibody (MAb 996) using RIA inhibition assay and characterized by IC50 values. At the same time, we have investigated the secondary structure of the compounds by circular dichroism and Fourier transform infrared spectroscopy in solution. Our data showed that the presence of D-amino acid residue(s) at position(s) 16P, 16PT17, or 16PTP18 resulted in gradually decreasing antibody binding, but the replacement of the L-Thr at position 19 almost abolished activity. Parallel with this reduction, changes in the conformer population have been detected. The propensity of the pTPTGTQ peptide to adopt folded, most probably beta-turn, structure in water can be in correlation with its essentially preserved antibody recognition. After further substitution, the peptide still contained beta- and/or gamma-turn folded secondary structural elements. The conformation of peptide ptptGTQ could be characterized mostly by semiextended (polyproline II) and probably classic gamma-turn conformers built up from D residues.     

Cyclic pentapeptides as models for reverse turns: determination of the equilibrium distribution between type I and type II conformations of Pro-Asn and Pro-Ala beta-turns

Cyclic pentapeptides are excellent models for reverse turns and have been used extensively in our laboratory to explore the influence of different amino acid sequences on turn preference. This paper is divided into two parts: In the first, we review our previous studies of cyclic pentapeptides. We summarize work that demonstrates the range of conformations possible within the cyclic pentapeptide backbone, the importance of sequence chirality in determining the backbone fold, and the utility of these cyclic pentapeptides as models for various turns. In the second, we present new results on two cyclic pentapeptides that contain beta-turns with Pro-Ala or Pro-Asn sequences in the i + 1 and i + 2 positions. By stereochemical criteria, a type I beta-turn is expected to be preferred by such L-L sequences. On the other hand, in proteins Asn occurs frequently in the i + 2 position of type II turns. We asked whether the same propensity would be manifest in an isolated model peptide, and if so, what the interactions were that influenced the relative stability of the type I and type II turns. To address these questions we have compared the conformational behavior of two peptides: cyclo(Gly-Pro-Ala-D-Phe-Pro) and cyclo(D-Ala-Pro-Asn-Gly-Pro). From previous studies, we anticipated that both peptides would contain an inverse gamma-turn and a beta-turn which consisted of either Gly-Pro-Ala-D-Phe or D-Ala-Pro-Asn-Gly in positions i to i + 3, respectively. Nuclear magnetic resonance analysis confirms this overall backbone conformation. Furthermore, quantitative nuclear Overhauser effect measurements in combination with molecular dynamics simulations and torsionally-forced energy minimizations have enabled us to determine that both type I and type II beta-turns are present in equilibrium in these peptides. The introduction of Asn in position i + 2 shifts this equilibrium significantly towards type II. We have done preliminary assessment of the possible side-chain/backbone conformations that contribute to the shift in populations.