Linear gramicidin activates neutrophil functions and the activation is blocked by chemotactic peptide receptor antagonist

The activation of functional responses in rabbit peritoneal neutrophils by gramicidin and the chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine methyl ester, was studied. Gramicidin activated superoxide generation, lysosomal enzyme release and a decrease in fluorescence of chlortetracycline-loaded cells, as for the chemotactic peptide. The maximum intensities of the responses by gramicidin were lower than that by chemotactic peptide. Responses by both these peptides could be inhibited by t-butyloxycarbonyl-methionyl-leucyl-phenylalanine, a chemotactic peptide receptor antagonist. Gramicidin gave responses at low doses comparable to that of the chemotactic peptide.

Measurement of FSH in the ovarian tissue by radioimmunoassay: Correlation to serum FSH levels and follicular development in the hamster

A method is described for monitoring the concentration of endogenous receptor-bound gonadotropin in the ovarian tissue. This involved development of a radioimmunoassay procedure, the validity of which for measuring all of the tissue-bound hormone has been established. The specificity of the method of measurement was indicated by the fact that high levels of FSH could be measured only in target tissue such as follicles, while non-target organs showed little FSH. Using this method, the amount of FSH in the non-luteal ovarian tissue of the hamster at different stages of the estrous cycle was quantitated and compared with serum FSH levels found at these times. No correlation could be found between serum and tissue FSH levels at all times. On the morning of estrus, for example, when the serum level of FSH was high, the ovarian concentration was low, and on the evening of diestrus-2 the ovary exhibited high concentration of FSH, despite the serum FSH concentration being low at this time. The highest concentration of FSH in the ovary during the cycle was found on the evening of proestrus. Although a large amount of this was found in the Graafian follicles, a considerable amount could still be found in the �growing� follicles. Ovarian FSH concentration could be considered to be a reflection of FSH receptor content, since preventing the development of FSH receptors by blocking initiation of follicular development during the cycle resulted in a decrease in the concentration of FSH in the ovary. The high concentration of FSH in the ovary seen on the evening of diestrus-2 was not influenced either by varying the concentration of estrogen or by neutralization of LH. Neutralization of FSH on diestrus-2, on the other hand, caused a drastic reduction in the ovarian LH concentration on the next day (i.e. at proestrus), thus suggesting the importance of FSH in the induction of LH receptors.

Synthetic peptide models for the redox-active disulfide loop of glutaredoxin. Conformational studies

Two cyclic peptide disulfides Boc-Cys-Pro-X-Cys-NHMe (X = L-Tyr or L-Phe) have been synthesized as models for the 14-membered redox-active disulfide loop of glutaredoxin. 'H NMR studies at 270 MHz in chloroform solutions establish a type I 0-turn conformation for the Pro-X segment in both peptides, stabilized by a 4-1 hydrogen bond between the Cys(1) CO and Cys(4) NH groups. Nuclear Overhauser effects establish that the aromatic ring in the X = Phe peptide is oriented over the central peptide unit. In dimethyl sulfoxide solutions two conformational species are observed in slow exchange on the NMR time scale, for both peptides. These are assigned to type I and type I1 p-turn structures with -Pro-Tyr(Phe)-as the corner residues. The structural assignments are based on correlation of NMR parameters with model 14-membered cyclic cystine peptides with Pro-X spacers. Circular dichroism studies based on the -S-Sn- u* transition suggest a structural change in the disulfide bridge with changing solvent polarity, establishing conformational coupling between the peptide backbone and the disulfide linkage in these systems.

Two levels of MCT1 and CD147 expression in the equine red blood cells and muscle

Monocarboxylate transporters (MCTs), especially the isoforms MCT1 - MCT4, cotransport lactate and protons across the cell membranes. They are thus essential for pH regulation and homeostasis in glycolytic cells such as red blood cells (RBCs), and skeletal muscle cells during intense exercise. In 70% of the Standardbred horses the lactate transport activity (TA) in RBCs is high and transport is mediated mainly by MCTs. In the rest 30% of the Standardbreds MCT mediated transport route is not active and the TA is low. MCTs need an ancillary protein for their proper localization and functioning in the plasma membrane. The ancillary protein for MCT1 and MCT4 is a member of immunoglobulin superfamily, CD147. Here we determined the expression of MCT isoforms and CD147 in equine RBCs and gluteal muscle. We sequenced the cDNA of horse MCT1 and CD147 to achieve horse-specific antibodies and to reveal sequence variations that may affect the TA of RBCs. The amount of MCT1 and CD147 mRNA in muscle were also studied. ---- In all, 73 horses representing different breeds were used. Blood samples were drawn from the jugular vein and muscle samples were taken either from gluteal muscle using biopsy needle or during castration from expendable cremaster muscle. The TA of RBCs was studied using radiolabeled lactate and the amount of MCT isoforms and CD147 in the plasma membranes using Western blotting. The level of mRNA in muscle cells was determined using qPCR. Isoforms MCT1 and MCT2 were found in the RBCs and isoforms MCT1 and MCT4 in the muscle cells of horses. The TA of RBCs was dependent on the expression of CD147 and MCT1 in the plasma membrane. Sequence variations were found in the cDNA of both MCT1 and CD147, but they did not explain the inactivity of MCT1 mediated transport route. The single nucleotide polymorphism (SNP) Met125Val in CD147 that existed parallel with an SNP in 3´-untranslated region explained, however, attenuation in CD147 expression in Standardbreds. A single mutation Ile51Val also decreased the expression of CD147 in one Warmblood. The MCT1 and CD147 mRNA concentrations in the gluteal muscle were higher in horses with higher MCT1 and CD147 expression in RBCs and lower in horses with minor expression of CD147 and MCT1. This suggests that the bimodal distribution of TA is due to differences in transcriptional regulation that is functioning in parallel in MCT1 and CD147 gene.

Mechanism of interaction of the antileukemic drug cytosine arabinoside with aromatic peptides : role of sugar conformation and peptide backbone

Interaction of the antileukemic drugs, cytosine-arabinoside (Ara-C) and adenosine-arabinoside (Ara-A) and a structural analogue, cytidine, with aromatic dipeptides has been studied by fluorescence and NMR spectroscopy. Ara-C and cytidine bind tryptophanyl and histidyl dipeptides but not tyrosyl dipeptides, while Ara-A does not bind to any of them. Both studies indicate association involving stacking of aromatic moieties. NMR spectra also indicate a protonation of the histidine moiety by Ara-C. In case of cytidine, the chemical shifts observed on binding to His-Phe imply that the backbone protons of the dipeptide participate in the binding. The conformation of the sugar and the base seem to play a very important role in the binding phenomenon as three similar molecules, Ara-C, Ara-A and cytidine bind in totally different ways.

Peptide models for b-turns.A circular dichroism study

The circular dichroism spectra of four 0-turn model peptides, Z-Aib-Pro-Aib-Pro- OMe (l), Piv-Pro-Aib-NHMe (2), Piv-Pro-D-Ala-NHMe (3) and Piv-Pro-Val-NHMe (4) have been examined under a wide range of solvent conditions, using methanol, hexafluoroisopropanol and cyclohexane. Type I and Type I1 0-turns have been observed for peptides 1 and 2 respectively, in the solid state, while the Pro-D-Ala sequence adopts a Type I1 Sturn in a related peptide crystal structure. A class C spectrum is observed for 1 in various solvents, suggesting a variant of a Type I(II1) structure. The Type I1 f3-turn is characterized by a CD spectrum having two positive CD bands at - 230 nm and - 202 nm, a feature observed in Piv-Pro- D-Ala-NHMe in cyclohexane and methanol and for Piv-Pro-Aib-NHMe in methanol. Peptide 2 exhibits solvent dependent CD spectra, which may be rationalized by considering Type 11, I11 and V reverse turn structures. Piv-Pro- Val-NHMe adopts nonaturn structures in polar solvents, but exhibits a class B spectrum in cyclohexane suggesting a population of Type I &turns.

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.

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