A helical peptide containing a majority of valyl residues. The crystal structure of t-butyloxy- carbonyl-(L-valyl-_-aminoisobutyryl)3-L-valyl methyl ester onohydrate

The monohydrate of the heptapeptide t-butyloxycarbonyl-(L-valyl-α-aminoiso-butyryl)3-L-valyl methyl ester crystallizes in the orthorhombic space group P212121 with four molecules in a unit cell with the dimensions α= 9.375, b = 19.413 and c = 25.878 ÅA. The structure has been solved by direct methods and refined to an R value of 0.059 for 3633 observed reflections. The molecule in the structure exists as a slightly distorted 310-helix stabilized by five 4 -> 1 intramolecular hydrogen bonds, indicating the overwhelming influence of α-aminoisobutyryl (Aib) residues in dictating helical fold even when a majority of residues in the peptide have a low intrinsic propensity to be in helices. Contrary to what is expected in helical structures, the valyl side chains, two of which are disordered, exhibit all three possible conformations. The molecules arrange themselves in a head-to-tail fashion along the c-axis. The columns thus generated pack nearly hexagonally in the crystal.

Conformation of a 16-residue zervamicin IIA analog peptide containing three different structural features: 310-helix, alpha helix and ß-bend ribbon

Boc-Trp-Ile-Ala-Aib-Ile-Val-Aib-Leu-Aib- Pro-Ala-Aib-Pro-Aib-Pro-Phe-OM(we here Boc is t-butoxycarbonyla nd Aib is a-aminoisobutyriac cid), a synthetica polar analog of the membrane-activefu ngal peptide antibioticz ervamtycinII A, crystallizesi n spaceg roupP 1 withZ =1 and cell parameters a = 9.086 ?0.002 A, b = 10.410 ?+ 0.002 A, c = 28.188 ? 0.004 A, a = 86.13 ? 0.01?, 13 = 87.90 ? 0.01?, and y = 89.27 ? 0.01?;o veralla greementf actorR = 7.3% for 7180 data (Fo > 3cr) and 0.91-A resolution. The peptide backbone makes a continuous spiral that begins as a 310-helix at the N-terminus, changes to an a-helix for two turns, and ends in a spiral of three fl-bends in a ribbon. Each of the fl-bends contains a proline residue at one of the corners. The torsion angles 4i range from -51? to -91? (average value -64o), and the torsion angles ai range from -1? to -46? (average value -31?). There are 10 intramolecularN H...OCh ydrogenb onds in the helix and two directh ead-to-taihl ydrogenb ondsb etween successive molecules. Two H20 and two CH30H solvent molecules fill additional space with appropriate hydrogen bonding in the head-to-tail region, and two additional H20 molecules form hydrogen bonds with carbonyl oxygens near the curve in the helix at Pro-10. Since there is only one peptide molecule per cell in space group P1, the molecules repeat only by translation, and consequently the helices pack parallel to each other.

Aqueous channels within apolar peptide aggregates. Solvated helix of Boc-Aib-Ala-Leu-Aib-Ala-Leu-Aib- -Ala-Leu-Aib-OMe. H2O.CH3OH

Although the peptide Boc-Aibl-Ala2-Leu3- Aib4-Alas Leu'-Aib7-Ala8-Leu9-Aib'0-OMe [with a t-butoxycarbonyl(Boc) blocking group at the amino terminus, a methyl ester (OMe) at the carboxyl terminus, and four a-aminoisobutyric (Aib) residues] has a 3-fold repeat of residues, the helix formed by the peptide backbone is irregular. The carboxyl-terminal half assumes an at-helical form with torsion angles ) and r of approximately -60° and -45°, respectively, whereas the amino-terminal half is distorted by an insertion of a water molecule between the amide nitrogen of Ala5 [N(5)] and the carbonyl oxygen of Ala2 [0(2)]. The water molecule W(1) acts as a bridge by forming hydrogen bonds N(5).W(1) (2.93 A) and W(1)---0(2) (2.86 A). The distortion of the helix exposes the carbonyl oxygens of Aib' and Aib4 to the outside environment, with the consequence that the helix assumes an amphiphilic character despite having all apolar residues. Neighboring helices in the crystal run in antiparallel directions. On one side of a helix there are only hydrophobic contacts with efficient interdigitation of leucine side chains with those from the neighboring helix. On the other side of the helix there are hydrogen bonds between protruding carbonyl oxygens and four water molecules that separate two neighboring helices. Along the helix axis the helices bind head-to-tail with a direct hydrogen bond N(2)-0(9) (3.00 A). Crystals grown from methanol/water solution are in space group P2, with a = 15.778 ± 0.004 A, b = 11.228 ± 0.002 A, c = 18.415 ± 0.003 A, = 102.10 ± 0.02ur and two formula units per cell for C49HON1003 2H2OCH3OH. The overall agreement factorR is 7.5% for 3394 reflections observed with intensities >3a(F), and the resolution is 0.90 A.

A possible role for a low molecular weight peptide in regulation of testosterone production by rat Leydig cells

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of Percoll purified Leydig cell proteins from 20- and 120-day-old rats revealed a significant decrease in a low molecular weight peptide in the adult rats. Administration of human chorionic gonadotropin to immature rats resulted in a decrease in the low molecular weight peptide along with increase in testosterone production. Modulation of the peptide by human chorionic gonadotropin could be confirmed by Western blotting. The presence of a similar peptide could be detected by Western blotting in testes of immature mouse, hamster, guinea pig but not in adrenal, placenta and corpus luteum. Administration of testosterone propionate which is known to inhibit the pituitary luteinizing hormone levels in adult rats resulted in an increase in the low molecular weight peptide, as checked by Western blotting. It is suggested that this peptide may have a role in regulation of acquisition of responsiveness to luteinizing hormone by immature rat Leydig cells.

PeptideMine - A webserver for the design of peptides for protein-peptide binding studies derived from protein-protein interactomes

Background: Signal transduction events often involve transient, yet specific, interactions between structurally conserved protein domains and polypeptide sequences in target proteins. The identification and validation of these associating domains is crucial to understand signal transduction pathways that modulate different cellular or developmental processes. Bioinformatics strategies to extract and integrate information from diverse sources have been shown to facilitate the experimental design to understand complex biological events. These methods, primarily based on information from high-throughput experiments, have also led to the identification of new connections thus providing hypothetical models for cellular events. Such models, in turn, provide a framework for directing experimental efforts for validating the predicted molecular rationale for complex cellular processes. In this context, it is envisaged that the rational design of peptides for protein-peptide binding studies could substantially facilitate the experimental strategies to evaluate a predicted interaction. This rational design procedure involves the integration of protein-protein interaction data, gene ontology, physico-chemical calculations, domain-domain interaction data and information on functional sites or critical residues. Results: Here we describe an integrated approach called ``PeptideMine'' for the identification of peptides based on specific functional patterns present in the sequence of an interacting protein. This approach based on sequence searches in the interacting sequence space has been developed into a webserver, which can be used for the identification and analysis of peptides, peptide homologues or functional patterns from the interacting sequence space of a protein. To further facilitate experimental validation, the PeptideMine webserver also provides a list of physico-chemical parameters corresponding to the peptide to determine the feasibility of using the peptide for in vitro biochemical or biophysical studies. Conclusions: The strategy described here involves the integration of data and tools to identify potential interacting partners for a protein and design criteria for peptides based on desired biochemical properties. Alongside the search for interacting protein sequences using three different search programs, the server also provides the biochemical characteristics of candidate peptides to prune peptide sequences based on features that are most suited for a given experiment. The PeptideMine server is available at the URL: http://caps.ncbs.res.in/peptidemine

C-H…O hydrogen bond mediated chain reversal in a peptide containing g-amino acid residue, determined directly from powder X-ray diffraction data

The finding that peptides containing -amino acid residues give rise to folding patterns hitherto unobserved in -amino acid peptides[1] has stimulated considerable interest in the conformational properties of peptides built from , and residues,[2] as the introduction of additional methylene (CH2) units into peptide chains provides further degrees of conformational freedom.

Probing the role of the C-H center dot center dot center dot O hydrogen bond stabilized polypeptide chain reversal at the C-terminus of designed peptide helices. Structural characterization of three decapeptides

The structural characterization in crystals of three designed decapeptides containing a double D-segment at the C-terminus is described. The crystal structures of the peptides Boc-Leu-Aib-Val-Xxx-Leu-Aib-Val- (D)Ala-(D)Leu-Aib-OMe, (Xxx = Gly 2, (D)Ala 3, Aib 4) have been determined and compared with those reported earlier for peptide 1 (Xxx = Ala) and the all L analogue Boc-Leu-Aib-Val-Ala-Leu-Aib-Val-Ala-Leu-Aib-OMe, which yielded a perfect right-handed a-helical structure. Peptides 1 and 2 reveal a right-handed helical segment spanning residues 1 to 7, ending in a Schellman motif with Ala(8) functioning as the terminating residue. Polypeptide chain reversal occurs at residue 9, a novel feature that appears to be the consequence of a C-(HO)-O-... hydrogen bond between residue 4 (CH)-H-alpha and residue 9 CO groups. The structures of peptides 3 and 4, which lack the pro R hydrogen at the C-alpha atom of residue 4, are dramatically different. Peptide 3 adopts a right-handed helical conformation over the 1 to 7 segment. Residues 8 and 9 adopt at conformations forming a C-terminus type I' beta-turn, corresponding to an incipient left-handed twist of the polypeptide chain. In peptide 4, helix termination occurs at Aib(6), with residues 6 to 9 forming a left-handed helix, resulting in a structure that accommodates direct fusion of two helical segments of opposite twist. Peptides 3 and 4 provide examples of chiral residues occurring in the less favored sense of helical twist; (D)Ala(4) in peptide 3 adopts an alpha(R) conformation, while (L)Val(7) in 4 adopts an alpha(L) conformation. The structural comparison of the decapeptides reported here provides evidence for the role of specific C-(HO)-O-... hydrogen bonds in stabilizing chain reversals at helix termini, which may be relevant in aligning contiguous helical and strand segments in polypeptide structures.

Diproline Templates as Folding Nuclei in Designed Peptides. Conformational Analysis of Synthetic Peptide Helices Containing Amino Terminal Pro-Pro Segments

The effect of N-terminal diproline segments in nucleating helical folding in designed peptides has been studied in two model sequences Piv-Pro-Pro-Aib-Leu-Aib-Phe-OMe (1) and Boc-Aib-Pro-Pro-Aib-Val-Ala-Phe-OMe (2). The structure of 1 in crystals, determined by X-ray diffraction, reveals a helical (RR) conformation for the segment residues 2 to 5, stabilized by one 4 -> 1 hydrogen bond and two 5 -> 1 interactions. The N-terminus residue, Pro(1) adopts a polyproline II (P-II) conformation. NMR studies in three different solvent systems support a conformation similar to that observed in crystals. In the apolar solvent CDCl3, NOE data favor the population of both completely helical and partially unfolded structures. In the former, the Pro-Pro segment adopts an alpha(R)-alpha(R) conformation, whereas in the latter, a P-II-alpha(R) structure is established. The conformational equilibrium shifts in favor of the P-II-alpha(R) structure in solvents like methanol and DMSO. A significant population of the Pro(1)- Pro(2) cis conformer is also observed. The NMR results are consistent with the population of at least three conformational states about Pro- Pro segment: trans alpha(R)-alpha(R), trans P-II-alpha(R) and cis P-II-alpha(R). Of these, the two trans conformers are in rapid dynamic exchange on the NMR time scale, whereas the interconversion between cis and trans form is slow. Similar results are obtained with peptide 2. Analysis of 462 diproline segments in protein crystal structures reveals 25 examples of the alpha(R)-alpha(R) conformation followed by a helix. Modeling and energy minimization studies suggest that both P-II-alpha(R) and alpha(R)-alpha(R) conformations have very similar energies in the model hexapeptide 1

X-ray studies on crystalline complexes involving amino acids and peptides. XXV. Structures of DL-proline hemisuccinic acid and glycyl-L-histidinium semisuccinate monohydrate and a comparative study of amino-acid and peptide complexes of succinic acid

DL-Proline hemisuccinic acid, C5H9NO2.1/2C4H6O4, M(r) = 174.2, P2(1/c) a = 5.254 (1), b = 17.480 (1), c = 10.230 (i) angstrom, beta = 119.60 (6)-degrees Z = 4, D(m) = 1.41 (4), D(x) = 1.42 g cm-3, R = 0.045 for 973 observed reflections. Glycyl-L-histidinium semisuccinate monohydrate, C8H13N4O3+.C4H5O4-.H2O, M(r) = 348.4, P2(1), a = 4.864 (1), b = 17.071 (2), c = 9.397 (1) angstrom, beta = 90.58-degrees, Z = 2, D(m) = 1.45 (1), D(x) = 1.48 g cm-3, R = 0.027 for 1610 observed reflections. Normal amino-acid and dipeptide aggregation patterns are preserved in the structures in spite of the presence of succinic acid/semisuccinate ions. In both the structures, the amino-acid/dipeptide layers stack in such a way that the succinic acid molecules/semisuccinate ions are enclosed in voids created during stacking. Substantial variability in the ionization state and the stoichiometry is observed in amino-acid and peptide complexes of succinic acid. Succinic acid molecules and succinate ions appear to prefer a planar centro-symmetric conformation with the two carboxyl (carboxylate) groups trans with respect to the central C=C bond. Considerable variation is seen in the departure from and modification of normal amino-acid aggregation patterns produced by the presence of succinic acid. Some of the complexes can be described as inclusion compounds with the amino acid/dipeptide as the 'host' and succinic acid/semisuccinate/succinate as the 'guest'. The effects of change in chirality, though very substantial, are not the same in different pairs of complexes involving DL and L isomers of the same amino acid.

Conformational studies on beta-bend containing a cis peptide unit

Conformational studies have been carried out on the X-cis-Pro tripeptide system (a system of three linked peptide units, in the trans-cis-trans configuration) using energy minimization techniques. For X, residues Gly, L-Ala, D-Ala and L-Pro have been used. The energy minima have been classified into different groups based upon the conformational similarity. There are 15, 20, 18 and 6 minima that are possible for the four cases respectively and these fall into 11 different groups. A study of these minima shows that, (i) some minima contain hydrogen bonds - either 4-->1 or 1-->2 type, (ii) the low energy minima qualify themselves as bend conformations, (iii) cis' and trans' conformations are possible for the prolyl residue as also the C(gamma)-endo and C(gamma)-exo puckerings, and (iv) for Pro-cis-Pro, cis' at the first prolyl residue is ruled out, due to the high energy. The available crystal structure data on proteins and peptides, containing cis-Pro segment have been examined with a view to find the minima that occur in solid state. The data from protein show that they fall under two groups. The conformation at X in X-cis-Pro is near extended when it is a non-glycyl residue. In both peptides and proteins there exists a preference for trans' conformation at prolyl residue over cis' when X is a non-glycyl residue. The minima obtained can be useful in modelling studies.

Identification of the active-site peptide of 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus oryzae

The non-oxidative decarboxylation of aromatic acids is a poorly understood reaction. The transformation of 2,3-dihydroxybenzoic acid to catechol in the fungal metabolism of indole is a prototype of such a reaction. 2,3-Dihydroxybenzoic acid decarboxylase (EC 4.1.1.46) which catalyzes this reaction was purified to homogeneity from anthranilate induced cultures of Aspergillus oryzae using affinity chromatography. The enzyme did not require cofactors like NAD(+), PLP, TPP or metal ions for its activity. There was no spectral evidence for the presence of enzyme bound cofactors. The preparation, which was adjudged homogeneous by the criteria of SDS-PAGE, sedimentation analysis and N-terminal analysis, was characterized for its physicochemical and kinetic parameters. The enzyme was inactivated by group-specific modifiers like diethyl pyrocarbonate (DEPC) and N-ethylmaleimide (NEM). The kinetics of inactivation by DEPC suggested the presence of a single class of essential histidine residues, the second order rate constant of inactivation for which was 12.5 M(-1) min(-1). A single class of cysteine residues was modified by NEM with a second order rate constant of 33 M(-1) min(-1). Substrate analogues protected the enzyme against inactivation by both DEPC and NEM, suggesting the Location of the essential histidine and cysteine to be at the active site of the enzyme. The incorporation of radiolabelled NEM in a differential labelling experiment was 0.73 mol per mol subunit confirming the presence of a single essential cysteine per active-site. Differentially labelled enzyme was enzymatically cleaved and the peptide bearing the label was purified and sequenced. The active-site peptide LLGLAETCK and the N-terminal sequence MLGKIALEEAFALPRFEEKT did not bear any similarity to sequences reported in the Swiss-Prot Protein Sequence Databank, a reflection probably of the unique primary structure of this novel enzyme. The sequences reported in this study will appear in the Swiss-Prot Protein Sequence Databank under the accession number P80402.

Crystal and molecular structure of Boc-Phe-Val-OMe; comparison of the peptide conformation with its dehydro analogue

The crystal structure of the peptide Boc-Phe-Val-OMe determined by X-ray diffraction methods is reported in this paper. The crystals grown from aqueous methanol are orthorhombic, space group P2(1)2(1)2(1), a = 11.843(2), b = 21.493(4), c = 26.676(4)Angstrom and V = 6790 Angstrom(3). Data were collected on a CAD4 diffractometer using MoK2 radiation (lambda = 0.7107 Angstrom) up to Bragg angle theta = 26 degrees. The structure was solved by direct methods and refined by a least-squares procedure to an R value of 6.8% for 3288 observed reflections. There are three crystallographically independent peptide molecules in the asymmetric unit. All the three molecules exhibit extended conformation. The sidechain of the Val(2) residue shows two different conformations. The conformation of the peptide Boc-Phe-Val-OMe is compared with the conformation of Ac-Delta Phe-Val-OH. It is observed that while Boc-Phe-Val-OMe exhibits an extended conformation, Ac-Delta Phe-Val-OH shows a folded conformation. The results of this comparison highlight the conformation constraining property of the Delta Phe residue. Interestingly, even though Boc-Phe-Val-OMe and Ac-Delta Phe-Val-OH are conformationally different, they exhibit similar packing patterns in the solid state. (C) Munksgaard 1995.

Observation of Water-Mediated Helix-Terminating Conformation in a Dehydrophenylalanine Peptide: Crystal and Solution Structure of the Octapeptide Ac-.DELTA.Phe-Val-.DELTA.Phe-Phe-Ala-Val-.DELTA.Phe-Gly-OMe

We have synthesised and determined the solution conformation and X-ray crystal structure of the octapeptide Ac-Delta Phe(1)-Val(2)-Delta Phe(3)-Phe(4)-Ala(5)-Val(6)-Delta Phe(7)-Gly(8)-OCH3 (Delta Phe = alpha,beta-dehydrophenylalanine) containing three Delta Phe residues as conformation constraining residues. In the solid state, the peptide folds into (i) an N-terminal (3)10(R)-helical pentapeptide segment, (ii) a middle non-helical segment, and (iii) a C-terminal incipient (3)10(L)-helical segment. The results of H-1 NMR data also suggest that a similar multiple-turn conformation for the peptide is largely maintained in solution. Though the C-terminal helix is incipient, the overall conformation of the octapeptide matches well with the conformation of the hairpins reported. Comparison of the pi-turn seen in the octapeptide molecule with those observed in proteins at the C-terminal end of helixes shows the structural similarity among them. A water molecule mediates the 5 --> 2 hydrogen bond in the pi-turn region. This is the first example of a water-inserted pi-turn in oligopeptides reported so far. Comparison between the present octapeptide and another (3)10(R)-helical dehydro nonapeptide Boc-Val-Delta Phe-Phe-Ala-Phe-Delta Phe-Val-Delta Phe-Gly-OCH3 solved by us recently, demonstrates the possible sequence-dependent conformational variations in alpha,beta-dehydrophenylalanine-containing oligopeptides.

Synthesis and Antioxidant Activity of Peptide-Based Ebselen Analogues

A series of di- and tripeptide-based ebselen analogues has been synthesized. The compounds were characterized by H-1, C-13, and Se-77 NMR spectroscopy and mass spectral techniques. The glutathione peroxidase (GPx)-like antioxidant activity has been studied by using H2O2, tert-butyl hydroperoxide (tBuOOH), and cumene hydroperoxide (Cum-OOH) as substrates, and glutathione (GSH) as a co-substrate. Although all the peptide-based compounds have a selenazole ring similar to that of ebselen, the GPx activity of these compounds highly depends on the nature of the peptide moiety attached to the nitrogen atom of the selenazole ring. It was observed that the introduction of a phenylalanine (Phe) amino acid residue in the N-terminal reduces the activity in all three peroxide systems. On the other hand, the introduction of aliphatic amino acid residues such as valine (Val) significantly enhances the GPx activity of the ebselen analogues. The difference in the catalytic activity of dipeptide-based ebselen derivatives can be ascribed mainly to the change in the reactivity of these compounds toward GSH and peroxide. Although the presence of the Val-Ala-CO2Me moiety facilitates the formation of a catalytically active selenol species, the reaction of ebselen analogues that has a Phe-Ile-CO2Me residue with GSH does not generate the corresponding selenol. To understand the antioxidant activity of the peptide-based ebselen analogues in the absence of GSH, these compounds were studied for their ability to inhibit peroxynitrite (PN)-mediated nitration of bovine serum albumin (BSA) and oxidation of dihydrorhodamine 123. In contrast to the GPx activity, the PN-scavenging activity of the Phe-based peptide analogues was found to be comparable to that of the Val-based compounds. However, the introduction of an additional Phe residue to the ebselen analogue that had a Val-Ala dipeptide significantly reduced the potency of the parent compound in PN-mediated nitration.