bluuebunny/crossref_metadata_2025 huggingface dataset:
Sequence and solution conformation of the 20‐residue peptaibols, saturnisporins SA II and SA IV
Authors: REBUFFAT SYLVIE , CONRAUX LAURENCE , MASSIAS MARCEL , AUVIN‐GUETTE CATHERINE , BODO BERNARD
Published: August 2010

Saturnisporins SA II and SA 1V are the major components of the 20‐residue peptaibol mixture isolated from a culture of the fungus Trichoderma saturnisporum. These peptides exhibit antibiotic activity against Staphylococcus aureus. Their sequences were derived from unequivocal methodology implying the combined use of positive ion FAB mass spectrometry and NMR: the majority of the sequences result from mass spectrometry fragmentations and the location of isomeric residues arises either from analysis of ROESY cross‐peaks between contiguous amide protons or from heteronuclear 2J or 3J 1H‐13C couplings detected in long‐range 1H‐13C COSY experiments. The sequence specific 1H and 13C NMR assignments are described. Saturnisporins SA II and SA IV exhibit similar secondary structures, as deduced from their ROESY patterns and 3JNHCαH coupling constant values, together with amide hydrogen‐deuterium exchange rates and temperature coefficients of amide and carbonyl groups. An overall α‐helical structure is proposed, maintaining two regions of distortion to this regular structure; i)the N‐terminal part, which contains 310 and mixed α‐310 turns, and ii) the Aib10‐Val15 region, including a Pro residue which accommodates a bend stabilized by two 310 H‐bonds.
Conformations of ψ[CH2NH] pseudopeptides
Authors: MA SOUGEN , SPATOLA ARNO F.
Published: August 2010

The cyclic pseudopentapeptide cyclo[Gly‐Proψ[CH2NH]Gly‐D‐Phe‐Pro] and its TFA salt were synthesized by solution methods, and their conformations were studied by NMR spectroscopy in both DMSO‐d6 and CDC13. While intramolecular hydrogen bonding is observed with some conformers, the nature of the solvent and the presence of TFA affects the relative structural rigidities of the compounds. No evidence was found for the ψ[CH2NH] or ψ[CH2NH+2] units acting as H donors in this series.
Resin immobilized synthetic peptides used to characterize phosphorylation and antigenic properties of insulin receptor autophosphorylation domains
Authors: CHAVANIEU ALAIN , CALAS BERNARD , GRIGORESCU FLORIN
Published: August 2010

To develop a common strategy in peptide design for kinase assay, antibody production and affinity purification, we investigated phosphorylation and antigenic properties of peptides immobilized on an aminated polyacrylic resin (Expansin ™) corresponding to autophosphorylation domains of the insulin receptor tyrosine kinase. Immobilized peptides (1143–1155) and peptide (1314–1330), designated pl 151 and p1322, respectively, were good substrates for the insulin receptor with Km, of 0.74 and 0.78 mM. By contrast, peptide (952–963), designated p960, was poorly phosphorylated. p1151 showed distinctive behaviour as a substrate, displaying a higher basal phosphorylation, a leftward shift of the insulin dose‐response curve (ED50= 0.7 ng mL‐ 1 insulin compared to 20 ng mL 1 for other substrates) and an inhibition by 90% of receptor autophosphorylation (ID50So = 0.5 mM). Similar substrate behaviour was observed with another tyrosine kinase, the pp60c‐src. Antibodies against P1151 and p1322 have comparable reactivity in ELISA, but the antibody against p960 was poor. While purified immunoglobulins (IgG) against both p1151 and p1322 were inhibitors of receptor autophosphorylation and kinase, in immunoprecipitation the IgG against p1151 mainly interacted with the phosphorylated receptor and that against p1322 with non‐phosphorylated forms. Functional mapping of the receptor with oligoclonal 1322‐antibody revealed inhibition of phosphate transfer to exogenous substrate poly(Glu,Tyr) (4:l) but not towards immobilized p1151. These data provide further support for the distinctive features of endogenous phosphorylation domain 1151. We conclude that immobilized peptides on polyacrylic resin offer a major new potential for use in kinase assays, immunization, immunoabsorbent techniques and purification of well defined oligoclonal antibodies.
Solid‐phase peptide synthesis on proteins
Authors: HANSEN PAUL ROBERT , HOLM ARNE , HOUEN GUNNAR
Published: August 2010

A new method for solid‐phase peptide synthesis in which a protein is used as the solid support has been developed. Two aspects of the method have been demonstrated. The peptides H‐Phe‐Leu‐Glu‐Glu‐Val‐OH (1) and H‐Leu‐Leu‐Ala‐Glj‐Val‐OH (2), respectively, were synthesized on the amino groups of BSA via a cleaveable linker, using the Fmoc group protecting scheme. The free peptides were obtained by cleavage from the protein with 95% TFA. precipitation in diethyl ether and additional work‐up by either dialysis or centrifugation through a membrane followed by gel filtration. The identity of the products was determined by amino acid analysis and HPLC. The peptide‐protein conjugates, H‐Ser‐Met‐Asp‐Thr‐Ser‐Asn‐Lys‐Glu‐Glu‐Lys‐BSA (3) and H‐Thr‐Val‐Leu‐BTG (4), were obtained in the same manner, omitting the cleavable linker group. It was found that 35‐50 peptide chains were conjugated per molecule BSA and BTG, respectively. Immunization of rabbits with conjugate 3 gave rise to peptide specific antibodies. This method will be useful for generation of sequence specific antibodies, since the peptide is conjugated to the carrier protein exclusively via its C‐terminus, and will allow synthesis of highly specific peptide‐protein conjugates.
Multiple column peptide synthesis, Part 2 (1, 2)
Authors: MELDAL MORTEN , HOLM CHARLOTTE BISGAARD , BOJESEN GUSTAV , JAKOBSEN MOGENS HAVSTEEN , HOLM ARNE
Published: August 2010

A manually operated apparatus for parallel multiple column soli‐phase peptide synthesis is described. It employs Fmoc‐amino acid‐O‐Dhbt or ‐Pfp esters in the continuous flow version of the polyamide method on small packed columns of kieselguhr supported resin in a reaction block of Teflon. The solvents and deprotecting reagents are dispensed from two washers in a parallel fashion and reagent consumption is low. Activated and protected amino acids are transferred from a dispenser tray as solutions, eight at a time. The use of the method is demonstrated by the synthesis of overlapping peptides from a protein structure and of analogous protease substrates. The products have been characterized by HPLC, FAB mass spectroscopy and amino acid analysis.
Disulfide linkage to polyacrylic resin for automated Fmoc peptide synthesis. Immunochemical applications of peptide resins and mercaptoamide peptides
Authors: MÉRY JEAN , GRANIER CLAUDE , JUIN MARIANICK , BRUGIDOU JEAN
Published: August 2010

The use of disulfide bonds for peptide–resin linkage in solid‐phase peptide synthesis was investigated using polyacrylic polymers (ExpansinTM) and automated Fmoc methodology. The disulfide moiety was bound to the support either by coupling a protected bifunctional handle or by an original stepwise procedure. Among the three different disulfide handles that were investigated, only the aminoethyldithio‐2‐isobutyric acid (AEDI) handle was stable enough to achieve peptide synthesis. A series of peptides of up to 10–20 amino acids were prepared in this manner, in good yield and purity. Rapid and quantitative peptide release was obtained by reduction with equimolecular amounts of dithiothreitol at pH 9 or tris(2‐carboxymethyl) phosphine at pH 4.5. This allowed direct and rapid coupling of the released cysteamide peptides to an activated protein carrier and the use of free or resin‐bound forms of the antigen in immunoassays.
Is an amphiphilic region responsible for the haemolytic activity of Bacillus thuringiensis toxin?†
Authors: SZABÓ ERIKA , MURVAI JÁNOS , FÁBIÁN PÉTER , FÁBIÁN FERENC , HOLLÓSI MIKLÓS , KaJTÁR JUDIT , BUZÁS ZSUZSA , SAJGÓ MIHÁLY , PONGOR SÁNDOR , ASBÓTH BENCE
Published: August 2010

The amino acid sequence of the 27 kDa protein responsible for the haemolytic activity of Bacillus thuringiensis subsp. israelensis toxin has been analysed by secondary structure prediction, helical wheel/net diagrams and molecular mechanics calculations. We found that segment 116–126 presumably forms a strongly amphiphilic α‐helix. This is supported by the findings that the synthesized segment 116‐126 (a) has a significant α‐helical content in water, and (b) displays an in vitro haemolytic activity comparable to that of bee venom peptide melittin. As segment 116‐126 is present in the haemolyzing, but not present in the non‐haemolyzing proteins from B. thuringiensis toxins, we suggest that this segment is responsible for the lytic potential of the B. thuringiensis subsp. israelensis protein.
Cyclic V3‐loop‐related HIV‐1 conjugate vaccines Synthesis, conformation and immunological properties
Authors: TOLMAN R.L. , BEDNAREK M.A. , JOHNSON B.A. , LEANZA W.J. , MARBURG S. , UNDERWOOD D.J. , EMINI E.A. , CONLEY A.J.
Published: August 2010

Branched undecapeptides with sequences related to the virus glycoprotein V3 domain sequences of the MN and IIIB variants of HIV‐1 were synthesized and cyclized with a peptide (amide) closure to cyclic decapeptides. Two‐dimensional NMR studies allowed protons for the MN variant‐related cycle (L‐697,250) to be assigned. Molecular modelling with distance geometry methods permitted a conformation to be identified which showed good agreement with ROESY and 2D NMR study data. A molecular dynamics simulation showed that the highly conserved loop tip sequence (Gly‐Pro‐Gly‐Arg) was in a conventional β‐turn less than 50% of the time. For evaluation of immunogenicity and antibody characterization studies, covalent carrier conjugates were prepared. 3‐Maleimidopropionylation of the Nle amino group of the cyclic peptides gave an electrophilic tether which captured a thiol group from a thiolated carrier protein, OMPC (outer membrane protein complex of Neisseria meningitidis). Through the use of a novel co‐conjugation procedure, soluble immunogen‐carrier molecules were prepared which had suitable physical properties for use as a vaccine. These V3‐loop‐based vaccines could elicit neutralizing antibody, but not consistently in all animals. Characterization of sera showed that responses were broadly virus neutralizing.
Vibrational spectroscopic studies of L,D‐alternating phenylalanine peptides
Authors: NAIK VAMAN M.
Published: August 2010

Infrared and Raman spectra of Boc‐(D‐Phe‐L‐Phe)n‐OMe, with n= 4 and 5, and Boc‐L‐Phe‐(D‐Phe‐L‐Phe)7‐OMe, have been obtained in crystalline and solution states. Based on the earlier spectroscopic studies on Boc‐(L‐Val‐D‐Val)n‐OMe, with n= 4, 6 and 8, and the predictions from the normal‐mode calculations of double‐stranded β‐helix conformations, structures have been assigned for the three oligopeptides. In the crystalline state, all three oligopeptides have a double‐stranded ↑↓β5,6 structure. Spectra from chloroform solution indicate that the solid‐state conformation is retained in this solvent for all three oligopeptides.
Solid‐phase synthesis of O‐mannosylated peptides: two strategies compared
Authors: ANDREWS D.M. , SEALE P.W.
Published: August 2010

A comparison of the O‐glycosylation of resin‐bound assembled peptides with the incorporation of glycosylated amino acids using established chemistry is presented. Fmoc/tert‐butyl‐based protecting groups were used for the peptidic moieties in conjunction with acetyl sugar protection. Koenigs‐Knorr glycosylations were carried out using protected bromomannose derivatives, the acceptor being threonine or serine, either in solution or within a resin‐bound peptide. The characterisation of microgram quantities of glycopeptides by the use of glycosidases in combination with mass spectrometry is also described.
Acyl, pseudotetra‐, tri‐ and dipeptide active‐core analogs of insect neuropeptides
Authors: NACHMANI RONALD J. , HOLMAN G. MARK , HAYES TIMOTHY K. , BEIER ROSS C.
Published: August 2010

Pseudopeptides of the achetakinin insect neuropeptide family were synthesized by replacing the amino acid blocks Phe‐, Phe‐Tyr‐, and Phe‐Tyr‐Pro‐ of the active‐core pentapeptide Phe‐Tyr‐Pro‐Trp‐GIy‐NH2 with hydrocinnamic acid, 6‐phenylhexanoic acid, and both 9‐phenylnonanoic and 6‐phenylhexanoic acid, respectively. All four of these analogs retained myotropic activity, demonstrating that the active core could be reduced from a pentapeptide to a modified dipeptide. Most notable of these was the pseudotetrapeptide hydrocinnamyl‐Tyr‐Pro‐Trp‐Gly‐NH2, which retained 70% of the potency and over 85% of the maximal activity of the parent pentapeptide. The N‐terminal amino group, the phenol ring of the Tyr residue, the sulfate moiety and the Gly residue of the insect sulfakinin active core Tyr(SO3H)‐Gly‐His‐Met‐Arg‐Phe‐NH2 were all replaced by dodecanedioic acid. The resulting pseudotetrapeptide, dodecandioyl‐His‐Nle‐Arg‐Phe‐NH2, elicited myostimulatory activity. Conversely, the related acyl pseudopentapeptide azelayl‐Gly‐His‐Nle‐Arg‐Phe‐NH2 proved myoinhibitory. A possible explanation for these disparate biological responses is discussed. These acyl pseudopeptides are important advances towards the eventual development of stable, potent mimetic agonists and antagonists of insect neuropeptides.
Systematic study of substance P analogs
Authors: WANG JIAN‐XIN , DIPASQUALEI ANGELA J. , BRAY ANDREW M. , MAEJI N. JOE , SPELLMEYER DAVID C. , GEYSEN H.MARIO
Published: August 2010

Recent developments in multiple peptide synthesis have made it feasible to synthesize and screen large numbers of peptide analogs simultaneously. We report here a model study of large scale screening of stereoisomers of substance P with systematic d‐aminO acid replacements. Such studies are useful in exploring conformational requirements of peptide‐receptor interaction and to provide empirical information for peptide drug design.

512 stereoisomers of SP were prepared by the multipin peptide synthesis method. Receptor binding affinities of these analogs were estimated by an iterative competition assay. Results obtained form a comprehensive database on the stereochemical requirement of SP binding to central NK1 receptor. Data from analogs with single d‐amino acid replacement are consistent with those previously reported showing that SP binding is highly sensitive to the chirality change of the C‐terminal residues (Gln6‐Leu10), but less sensitive to the chirality change of the N‐terminal residues (Arg1‐Gln5). A qualitative analysis of the database by comparison of series of peptide pairs revealed a repeated pattern of affinity change with d‐amino acid replacement, suggesting a largely additive binding activity of SP from each residue. On the other hand, possible intramolecular interactions between some N‐terminal and C‐terminal residues to form an optimal binding conformation were also found.

A set of 189 peptides with IC50 values less than 5 μM was subjected to an empirical QSAR analysis using a linear additive model. The relative contribution coefficients obtained agreed with the observation that the predominant contribution comes from the C‐terminal residues, suggesting considerable independency of each residue on binding to NK1 receptors. However, the possibility of interactions among residues in forming an optimal binding conformation can not be excluded as implied by a marginal fitting of the data to this model (R2= 0.61). The poor prediction of binding activity by the model used emphasizes the importance of systematic, empirical approaches in discovery and designing new peptide analogs, which is now achievable using multipeptide synthesis techniques.
α‐Amylases from Thermoactinomyces vulgaris: characteristics, primary structure and structure prediction
Authors: HANSEN GERD , HEESE OLAF , HÖHNE WOLFGANG E. , HOFEMEISTER BRIGITTE
Published: August 2010

Two amylolytic active protein fractions (named α‐amylase 1 and α‐amylase 2) were isolated from the bacterium Thermoactinomyces vulgaris strain 94‐2A. α‐Amylase 1 had a molecular mass of 51.6 kDa, whereas α‐amylase 2 consists of two fragments which have molecular masses of 17.0 and 34.6 kDa, respectively. These two fragments are products from a proteolytic cleavage of a‐amylase 1 at amino acid position 303 (tryptophan) by a serine protease (thermitase) which is also produced by T. vulgaris. The purified α‐amylase 1 and 2 follow the Michaelis‐Menten kinetics in the presence of starch as substrate with Km values of 1.37 ± 0.07 and 1.29 ± 0.18 mg/mL, respectively. In effect they differ in their stability characteristics. The amino acid sequence of α‐amylase from T. vulgaris derived from DNA sequence (1) was compared with those of other α‐amylases. It reveals high homologies to α‐amylases from other microorganisms (e.g. B. polymyxa, A. oryzae, S. occidentalis and S.fibuligera). A three‐dimensional structure model for α‐amylase 1 on the basis of the 3 Å X‐ray structure of Taka‐amylase was constructed.
Purification and characterization of gonadotropin I and II from pituitary glands of tuna (Thunnus obesus)
Authors: OKADA TSUYOSHI , KAWAZOE ICHIRO , KIMURA SHOJI , SASAMOTO YASUHIKO , AIDA KATSUMI , KAWAUCHI HIROSHI
Published: August 2010

The duality of teleost pituitary gonadotropins was established in an advanced marine fish, the tuna (Thunnus obesus). Two different molecular forms of gonadotropins, designated tGTH I and tGTH II, were isolated from an alcoholic extract of pituitary glands following ion‐exchange chromatography and reversed‐phase HPLC. Both tGTH I and tGTH II stimulated estradiol‐17β and testosterone production in tuna ovarian follicles in vitro, although responses to tGTH II were significantly greater than those to tGTH I. Each gonadotropin consisted of α‐ and β‐subunits. tGTH I was stable in acidic conditions, whereas tGTH II dissociated into two subunits after acid treatment. Alpha subunits of tGTH I and tGTH II had identical amino acid sequences of 94 amino acid residues. The tGTH Iβ and tGTH IIβ consisted of 102 and 115 amino acid residues, respectively, and showed 35% sequence identity. tGTH Iβ is structurally more similar to salmon GTH Iβ than to salmon GTH IIβ, whereas tGTH IIβ is more similar to salmon GTH IIβ. Thus it is evident that the tuna pituitary gland produces two chemically distinct gonadotropins.
Harnessing D‐amino acids for peptide motif designs
Authors: BOBDE VIVEK , SASIDHAR Y.U. , DURANI SUSHEEL
Published: August 2010

In examining the use of d‐amino acids in designing specific peptide folding motifs, the tetrapeptide Boc‐d‐Glu‐Ala‐Gly‐Lys‐NHMe 1 and its analog 2 featuring l‐Glu were synthesized for a comparison of their solution conformations by NMR spectroscopy. The temperature coefficients of amide proton resonances, NOE data, side‐chain CH2 anisotropies and salt titration results suggest a weak type II reverse‐turn conformation for peptide 2, and a tandem type II’ turn‐310‐helix conformation of appreciable conformational stability for peptide 1 in apolar solvents. The latter is of potential interest as the N‐terminal helix cap that could support the design of longer 310 helices. Possible origins of appreciable difference in the conformational stabilities of the diastereomers are discussed.
NMR spectra and restrained moleclar dynamics of the mushroom toxin viroisin
Authors: BHASKARAN R. , YU CHIN
Published: August 2010

The conformation of viroisin, the monocyclic toxic heptapeptide of the virotoxin family from mushroom Amanita virosa, was analysed using two‐dimensional nuclear magnetic resonance spectroscopy and restrained molecular dynamics simulations. All proton signals were completely assigned, and interproton distances were determined using data from rotating‐frame nuclear Overhauser enhancement. The backbone dihedral angles were deduced from measurements of coupling constants. The temperature dependence of the amide proton chemical shifts provided information about hydrogen bonding. Six probable solution conformations of viroisin were derived from the use of distance geometry and restrained molecular dynamics based on a set of distance constraints obtained from experimental data. The results of the structural analysis indicate that viroisin has a well ordered conformation in solution. In all these conformations the functional groups essential for toxicity orient themselves in the same direction so as to bind to the target proteins. This feature is consistent with previous results about the formation of a hydrophobic pocket on one side of the molecule.
Solid‐phase syntheses of phosphorylated and thiophosphorylated peptides related to an EGFR sequence
Authors: TEGGE W.
Published: August 2010

The 13 amino acid EGFR‐sequence AENAEYLRVAPQS‐NH2 containing the in vivo autophosphorylated Tyr 1171, was synthesized by Fmoc continuous‐flow SPPS with and without N‐terminal Boc protection. In addition to the native sequence, peptides in which tyrosine was exchanged by serine and threonine were prepared. Global phosphorylation of the unprotected hydroxyl amino acids on the resin with di‐tert‐butyl‐N,N‐diethylphosphoramidite and 1H‐tetrazole followed by in situ oxidation of the resulting phosphites with tert‐butyl hydroperoxide or with dibenzoyl tetrasulfide resulted in the tyrosine‐, serine‐ and threonine‐phosphorylated and ‐thiophosphorylated sequences, respectively. The quality of the products after phosphorylation with N‐terminal protection was better than without. Whereas the serine‐ and threonine‐thiophosphate group was stable, tyrosine‐thiophosphate turned out to be hydrolytically labile under acidic conditions. The rate of hydrolysis was determined with the tyrosine‐thiophosphorylated model dipeptide Ac‐Tyr‐Gly‐OH between pH 0.1 and 8. Hydrolysis was fastest at pH 3, with a half‐time of 12.5 h at room temperature. The tyrosine‐thiophosphate group was completely stable at pH 8.
Leiurotoxin I, a scorpion toxin specific for Ca2+‐activated K+ channels Structure‐activity analysis using synthetic analogs
Authors: SABATIER J.‐M. , FREMONT V. , MABROUK K. , CREST M. , DARBON H. , ROCHAT H. , RIETSCHOTEN J. VAN , MARTIN‐EAUCLAIRE M.‐F.
Published: August 2010

Recently, we reported a structure‐activity relationship study on P05, a novel leiurotoxin I‐like scorpion toxin which is selective for the apamin‐sensitive Ca2+‐activated K+channel [Sabatier et al. (1993) Biochemistry32, 2763–27701. Arg6, Arg7 and C‐terminal His31 appeared to be key residues for P05 biological activity. Owing to the high sequence identity between P05 and leiurotoxin I (87%), several analogs of leiurotoxin I (Lei‐NH2) with point mutations at these positions were designed and chemically synthesized using an optimized solid‐phase technique. The synthesized peptides were [L6]Lei‐NH2, [R7]Lei‐NH2, Lei‐OH and [R7]Lei‐OH, as well as fragment [R7,Abu8]N4‐S11‐NH2. A chimeric analog ([M22,K24,R27]Lei‐NH2), which possesses part of the iberiotoxin C‐terminus, was also constructed. Circular dichroism analyses of these analogs, in agreement with their structural models obtained by molecular dynamics, showed that the point mutations did not significantly affect the overall secondary structures, as compared to natural Lei‐NH2. All the peptides and natural toxins were compared in vitro for their capacity to inhibit binding of [125I]‐apamin to rat brain synaptosomes, and in vivo for their specific neurotoxicity in mice. The Arg6 residue was essential for high biological activity of leiurotoxin I. Further, substitution of Met7 in the natural toxin by Arg7, or C‐terminal amidation of His31, greatly increased affinity for the apamin receptor but did not significantly affect toxin neurotoxicity. Remarkably, the chimeric analog [M22,K24,R27]Lei‐NH2 was found to retain leiurotoxin I‐like activity, thus indicating that the negatively charged residues Asp24, and Glu27 (and Ile22) are not directly involved in the high toxin bioactivity. However, the chimeric molecule had no iberiotoxin‐like effect on rat muscular maxi‐K channels incorporated in lipid bilayers.
Migration of monocytes in the presence of elastolytic fragments of elastin and in synthetic derivates Structure‐activity relationships
Authors: BISACCIA F. , MORELLI M.A. CASTIGLIONE , BIASI M. DE , TRANIELLO S. , SPISANI S. , TAMBURRO A.M.
Published: August 2010

YGVG and GLVPG, two new chemokinetic peptides, were identified in elastolytic digests of elastin, besides the known chemoattractant hexapeptide VGVAPG. In order to identify possible sequences responsible for the chemotactic and chemokinetic activities and to obtain structure‐activity relationships we synthesized some analogues of these peptides: FGVG (an analogue of YGVG), GVAPG and VGAPG (derived from the hexapeptide by deletion of Val1 or Val3). FGVG has a higher chemotactic activity than YGVG (chemotactic indices of 0.62 and 0.49, respectively, at 10−11 M) and is both chemotactic and chemokinetic.

Checkerboard analysis demonstrated that both peptides derived from the hexapeptide present, in addition to the chemotactic activity, a cheniokinetic activity. The chemotactic index of GVAPG is 0.66 at 10−10M, while for VGAPG it is 0.86 at 10−9 M.

These results indicate that the deletion of the N‐terminal residue of the elastin chemotactic peptides, VGVAPG and GFGVG, gives rise to chemokinetic activity.

CD and NMR studies showed that all peptides are largely unordered in aqueous solution. © Munksgaard 1994.
Conformational stability of the endothelin/sarafotoxin family of peptides
Authors: ATKINS A.R. , RALSTON G.B. , SMITH ROSS
Published: August 2010

There are significant differences between the structures reported for members of the endothelin/sarafotoxin family of peptides, but also for the same peptides studied by different groups, raising the possibility that some of the differences are attributable to variation in solution conditions rather than intrinsic structural heterogeneity. We have shown, using circular dichroism spectroscopy and equilibrium sedimentation, that the secondary structures of these peptides are little affected by wide variations in pH, or by self‐association. Although acetonitrile has a pronounced effect on the extent of peptide self‐association it does not appear to alter the backbone structure of sarafotoxin SRTb, and has only minor effects on endothelin‐1 andendothelin‐3. The observed conformational variation thus appears largely to reflect sequence‐dependent differences. © Munksgaard 1994.
Structure‐activity studies on the vasoactive intestinal peptide pharmacophore
Authors: BOLIN DAVID R , COTTRELL JEANINE , GARIPPA RALPH , MICHALEWSKY JOSEPH , RINALDI NANCY , SIMKO BEVERLY , O'DONNELL MARGARET
Published: August 2010

From previous work, the primary functional groups, i.e. side chains, of the vasoactive intestinal peptide which are responsible for interaction with the VIP receptor have been identified. One of these sites, the side chain of tyrosine22 is essential for high receptor affinity. The present work aims to examine this site in greater detail. Several Boc‐substituted‐phenylalanine derivatives were prepared and incorporated into VIP analogs as replacement for tyrosine22. These analogs, of the form Ac‐[Lys12,Nle17,X22,Va126,Thr28]‐VIP, were assayed as smooth muscle relaxants and found to be full agonists of native VIP. Most of the analogs, however, proved to be less potent than the parent analog by up 00 300‐fold. A few analogs, all possessing electron‐donating substituents, retained nearly full potency. Two compounds, 3‐F,4‐OH‐Phe, 42 and 3‐OCH3,4‐OH‐Phe, 43, were found to be 1.5‐ and 3.4‐fold more potent than the parent compound, which equates to being 8.9‐ and 20‐fold more potent than native VIP. Compound 43 was also found to be active as a bronchodilator in vivo in guinea pigs, with slightly over 2‐fold enhanced potency and a significantly longer duration of action (>20min) when compared to the parent compound (5 min). The physical characteristics of the various substituents and their effect on biological activity are discussed with a brief analysis by QSAR techniques.
A sensitive novel staining agent for the resolved proteins on PAGE
Authors: ALI RASHDA , SAYEED SYED ASAD , KHAN AHMAD ALI
Published: August 2010

A more sensitive, stable and fast‐reacting dye was isolated from the bark of Ratanjot by a simple solvent extraction procedure at high pH. The dye produced sharp, clear distinct stable bands of known proteins when resolved on polyacrylamide gel electrophoresis (PAGE) using sodium dodesyl sulfate (SDS), showing a rapid and strong affinity with protein molecules. Not only did proteins from peanut, separately stained with the new dye and Coomassie Brilliant Blue R‐250, produce rapid comparable results, but also the protein bands were easily visualized and were less diffusible with clearer boundaries in the case of the new staining agent. © Munksgaard 1995.
Incorporation of thioamide linkages into a growing peptide under SPPS conditions improved by salt effects
Authors: LE HOANG‐THANH , MAYER MICHEL , THORET SYLVIANE , MICHELOT ROBERT
Published: August 2010

Amino acid methyl dithioesters may be coupled in the presence of DMAP and salts to a growing peptide chain on a polymeric resin with high coupling yields and low racemization. © Munksgaard 1995.
Studies on conformational consequences of i to i+ 3 side‐chain cyclization in model cyclic tetrapeptides
Authors: RAO MAREPALLI H. , YANG WEI , JOSHUA HENRY , BECKER JEFFREY M. , NAIDER FRED
Published: August 2010

In an effort to explore the effect of ring size on the biologically active conformation of cyclic analogs of the mating pheromone α‐factor (WHWLQLKPGQPMY) from Saccharomyces cerevisiae, eight cyclic tetrapeptides corresponding to the KPGQ portion of α‐factor were synthesized. These N‐α‐acetyl/carboxyl amide terminal cyclic tetrapeptides were prepared on a 4‐methylbenzhydrylamine resin using orthogonal Boc, Frnoc, OFm and OtBut protecting groups and HOBt‐DIPC accelerated active esters or urethane‐protected N‐carboxyanhydrides. On‐resin cyclization of the side‐chain amino and carboxyl groups of the first and fourth residues, respectively, was performed with the BOP reagent to generate lactams containing 14–18 atoms. HF cleavage resulted in two products, the desired cyclic tetrapeptide and a major side product. All peptides were purified to near homogeneity (>99%) by using reversed‐phase HPLC and were characterized by FABMS and 1H NMR. Certain constrained cyclic tetrapeptides appear to be a mixture of isomers at room temperature as evidenced by HPLC and NMR. The major side product has been identified as a cyclo dimer, obtained as a consequence of interchain cyclization on the resin. CD analysis in several solvents gives evidence that some of the cyclic tetrapeptides exist in β‐turn conformations. © Munksgaard 1995.
Structure–activity relationship study of a scorpion toxin with high affinity for apamin‐sensitive potassium channels by means of the solution structure of analogues
Authors: INISAN A.G. , MEUNIER S. , FEDELLI O. , ALTBACH M. , FREMONT V. , SABATIER J.‐M. , THÉVAN A. , BERNASSAU J.‐M. , CAMBILLAU C. , DARBON H.
Published: August 2010

Scorpion venoms contain numerous toxic polypeptides displaying various pharmacological activities. These toxins interact with ion channels of excitable membranes. Long toxins (60–70 amino acids) are known to interact with sodium channels, whereas most of the short toxins (31–37 amino acids) found their toxicity in modifying the potassium channel functions. A family of short scorpion toxins are known to interact specifically with apamin‐sensitive calcium‐activated potassium channels. Structure‐activity relationship studies of these toxins have demonstrated that a short region located on the solvent‐exposed side of an α‐helix is involved in the interaction with their receptor. Two positions, i.e. residues 6 and 7 in the sequence, are essential for the full activity of these molecules. We have synthesized analogues of these toxins and demonstrated that the three‐dimensional structure is not affected by these mutations, and thus that the observed variations of activity are only due to the chemical function carried by the side chain. This interaction between the toxins and their receptor is thus purely electrostatic. © Munksgaard 1995.
A new neurohypophysial peptide, seritocin ([Ser5,Ile8]‐oxytocin), identified in a dryness‐resistant African toad, Bufo regularis
Authors: CHAUVET JACQUELINE , MICHEL GILLES , OUEDRAOGO YOUSSOUFOU , CHOU JAMES , CHATT BRIAN T. , ACHER ROGER
Published: August 2010

From the pituitary neurointermediate lobe of the African toad Bufo regularis, vasotocin, hydrin 2 (vasotocinyl‐Gly) and a mesotocin‐like peptide have been isolated by HPLC and characterized by mass spectrometry, amino acid sequence and chromatographic coelution with synthetic peptides. The mesotocin‐like peptide has been identified as [Ser5,Ile8]‐oxytocin in place of mesotocin ([Ile8]‐oxytocin) found in all other amphibians investigated to date. The name seritocin is suggested. The molecule is virtually devoid of oxytocic activity on rat uterus in contrast to mesotocin. On the other hand, the molar ratio of hydrin 2 to vasotocin in the pituitary reaches 2, whereas it is about 1 in toads and frogs from temperate regions. B. regularis is an anuran species able to withstand a hot and dry season by burrowing. The possible relationship between occurrence of seritocin and Actaptation to arid environment remains to be demonstrated. © Munksgaard 1995.
Chemical synthesis and characterization of ShK toxin: a potent potassium channel inhibitor from a sea anemone
Authors: PENNINGTON M. W. , BYRNES M. E. , ZAYDENBERG I. , KHAYTIN I. , DE CHASTONAY J. , KRAFTE D. S. , HILL R. , MAHNIR V. M. , VOLBERG W .A. , GORCZYCA W. , KEM W. R.
Published: August 2010

ShK‐toxin, a 35 residue peptide isolated from the sea anemone Stichodactyla helianthus, was synthesized using an Fmoc strategy and successfully folded to the biologically active form containing three intramolecular disulfide bonds. The ability of synthetic ShK toxin to inhibit specific [125I]‐dendrotoxin I binding to rat brain membranes slightly exceeded (was more potent than) that of the natural ShK toxin sample, but was comparable with previously reported data for ShK toxin. The peptide toxin inhibited [125I]‐charybdotoxin binding to Jurkat T lymphocytes with an IC50 value of 32 pM. In addition, Jurkat T lymphocytes Kv1.3 potassium channels were inhibited with an IC50 value of 133 PM. Owing to their unique structure and high affinity for at least some potassium channels, ShK toxin and related sea anemone potassium channel toxins may become useful molecular probes for investigating potassium channels. © Munksgaard 1995.
Identification and characterization of a novel synthetic peptide substrate specific for Src‐family protein tyrosine kinases
Authors: LAM KIT S. , WU JINZI , LOU QIANG
Published: August 2010

Using a random combinatorial peptide library method [Wu, J., Ma, Q. N. & Lam, K. S. (1994) Biochemistry33, 14825–14833] a novel peptide, YIYGSFK, was identified as a substrate for p60c‐src protein tyrosine kinase. Mass spectrometric analysis showed that tyrosine‐3 from the N‐terminus was the phosphorylation site. Kinetic studies showed that the Km of YIYGSFK for p60c‐src was 55 μM, about 6.4‐fold lower than a peptide derived from p34cdc2 [cdc2(6–20), KVEKIGEGTYGVVYK], which had been reported to be a specific and efficient substrate for the Src‐family protein tyrosine kinases. Comparison of the specificity of YIYGSFK and cdc2(6–20) as a substrate for various Src‐family and non‐Src‐family protein tyrosine kinases suggests that YIYGSFK is a much more specific and efficient substrate for the Src‐family protein tyrosine kinases. © Munksgaard 1995.
Determination of the ratios of the aromatic amino acid residues by first‐ or second‐derivative UV spectrometry for a simple characterization of peptides
Authors: MICLO LAURENT , PERRIN EMMANUEL , DRIOU ALAIN , MELLET MICHEL , LINDEN GUY
Published: August 2010

A method for the simultaneous determination of the ratios of the three aromatic amino‐acid residues in peptides was set up in acidic conditions. Binary and ternary mixtures of these amino acids were prepared, and first‐ and second‐derivative spectra then calculated from their 0.1 nm resolution spectra between 240 and 320 nm. Certain spectral bands were chosen to differentiate tyrosine from tryptophan on the first‐derivative spectra, and phenylalanine from tyrosine and tryptophan on the second‐derivative spectra. Variation of the amplitude of the chosen bands was shown to be a linear function of the ratio of the aromatic amino acids in the mixture. This technique was validated with peptides whose sequence was known. The difference between theoretical and experimentally determined ratios was lower than 10%. Since the results are obtained as ratios, neither the concentration nor the nature of the peptide has to be known. The feasibility of application using a photodiode array detector with high resolution in reversed‐phase high‐performance liquid chromatography is discussed. © Munksgaard 1995.
Role of ring size on the secondary structure and antibiotic activity of gramicidin S
Authors: TAMAKI MAKOTO , ARAI IZUMI , AKABORI SADATOSHI , MURAMATSU ICHIRO
Published: August 2010

In order to investigate the contribution of ring size to the secondary structure and antibiotic activity of gramicidin S, many analogs consisting of 6, 7, 8, 9, 11, 12, 13, and 14 amino acid residues were synthesized. In the analogs with smaller ring sizes than that of gramicidin S, only des‐Val1‐, des‐Leu3‐ and des‐Pro5‐gramicidin S showed weak activity against the gram‐positive micro‐organisms tested. On the other hand, all analogs having larger rings, in which L‐ or D‐Leu residues were inserted, were active. The activity of the analogs consisting of 10, 11 and 12 amino acid residues was stronger than those of the analogs with other ring sizes, and the activity of endo‐Leu2a‐gramicidin S against S. epidermidis SP‐al‐1 and B. subtilis ATCC 6633 was twice of that of gramicidin S.
Designed polyanionic coiled‐coil proteins: acceleration of heparin cofactor II inhibition of thrombin
Authors: MELTON LAURA G. , CHURCH FRANK C. , ERICKSON BRUCE W.
Published: August 2010

Novel polyanionic proteins were designed to increase the rate of heparin cofactor II (HC) inhibition of α‐thrombin, an essential protease in the coagulation cascade. Two α‐helical coiled‐coil proteins, a 62‐residue dimer containing 8 Glu residues (E8C) and a 104‐residue dimer containing 14 Glu residues (E14C), plus two 31‐residue control peptides containing 8 Glu residues each (E8A and E8B), were chemically synthesized, structurally characterized and enzymatically assayed. Circular dichroic spectrophotometry indicated that both E8C and E14C formed stable two‐chain α‐helical coiled coils at pH 7 and 25 °C. The control peptides were only partially α‐helical. E14C remained folded at 90 °C but E8C was half unfolded at 49 °C. Coiled‐coil proteins E8C and E14C maximally accelerated by 35‐ and 33‐fold, respectively, the rate of HC inhibition of α‐thrombin. None of these compounds accelerated antithrombin inhibition of α‐thrombin, and neither control peptide accelerated HC inhibition of α‐thrombin. Acceleration of the HC inhibition of α‐thrombin showed bimodal dependence on the concentration of the polyanionic protein, which is consistent with formation of a HC‐coiled‐coil‐thrombin ternary complex. The results suggest that antithrombotic polyanionic α‐helical coiled‐coil proteins can be designed and synthesized and that the occurrence of secondary structure can be correlated with biologcal activity. © Munksgaard 1995.
Studies on the neuroexcitotoxin β‐N‐oxalo‐L‐α, β‐diaminopropionic acid and its isomer α‐N‐oxalo‐L‐α, βdiaminopropionic acid from the root of Panax species
Authors: LONG YI‐CHENG , YE YUN‐HCA , XING QI‐YI
Published: August 2010

The neuroexcitotoxic nonprotein amino acid, β‐N‐oxalo‐L‐α, β‐diaminopropionic acid (β‐N‐ODAP) was found in Panax species such as Panax ginseng C.A. Meyer’(cultivated in Northeastern China), Panax quinquefolius (L), Panax notoginseng ‘F.H. Chen’(cultivated in Southwestern China), Korean red ginseng and Jilin (China) red ginseng. β‐N‐ODAP was verified to be a natural substance, and its isomer α‐N‐oxalo‐L‐α, β‐diaminopropionic acid (α‐N‐ODAP) is very likely an artifact generated during the separation process. The interconversion between β‐N‐ODAP and α‐N‐ODAP was observed. Results showed that both β‐N‐ODAP and α‐N‐ODAP can easily be converted into their isomers at higher temperature or in acidic and basic solution. Their interconversion equilibrium constant and rate constants were obtained. Upon extensive study and a good understanding of the interconversion, accurate physical constants and spectroscopic data such as MS, 1H NMR, 13C NMR and UV Mere obtained. ©Munksgaard 1996.
α‐Kirilowin, a novel ribosome‐inactivating protein from seeds of Trichosanthes kirilowii (family Cucurbitaceae): a comparison with β‐kirilowin and other related proteins
Authors: WONG RICKY NGOK SHUN , DONG TING XIA , NG TZI BUN , CHOI WAI TO , YEUNG HIN WING
Published: August 2010

A novel ribosome‐inactivating protein (RIP) designated α‐kirilowin was isolated from the seeds of Trichosanthes kirilowii. The molecular weight of α‐kirilowin was estimated by SDS‐polyacrylamide gel electrophoresis to be 28 800 Da, which is slightly larger than another previously characterized ribosome‐inactivating protein, β‐kirilowin. The amino‐acid composition of α‐kirilowin grossly resembled β‐kirilowin and other ribosome‐inactivating proteins isolated from T. kirilowii tissues, including trichokirin, trichosanthin and karasurin. Intense immunological cross‐reactivity between the two kirilowins was detected by immunodiffusion. The N‐terminal sequence of α‐kirilowin was identical to that of β‐kirilowin, at least in the first ten residues. Peptide fingerprinting indicated both kirilowins were closely related. Biological activities as determined by inhibition of protein synthesis in a cell‐free system, suppression of [3H]‐thymidine incorporation into mouse melanoma cells and induction of abortion in mice were very similar for both kirilowins. We propose that the size difference between α‐and β‐kirilowin is either due to a C‐terminal extension in α‐kirilowin or differences in glycosylation, or a combination of both. ©Munksgaard 1996.
The entire γ‐carboxyglutamic acid‐ and helical stack‐domains of human coagulation factor IX are required for optimal binding to its endothelial cell receptor
Authors: PROROK MARY , GENG JIE‐PING , WARDER SCOTT E. , CASTELLINO FRANCIS J.
Published: August 2010

The minimal region of the γ‐carboxyglutamic acid (Gla) domain of human factor (f) IX that interacted with its putative bovine aortic endothelial cell (BAEC) receptor was examined by chemical synthesis of peptides with sequence counterparts in this region of the protein, and assessment of their relative abilities to compete with fIX for receptor binding. We found that IC50 values (total peptide concentrations needed to achieve 50% inhibition of binding of [125I]‐fIX to BAEC) were ca. 18 nM for unlabeled fIX and 23 nM for the peptide consisting of the entire Gla domain/helical stack (HS) region (residues 1–47) of fIX. The peptide containing only the Gla domain of fIX (residues 1‐38) displayed an IC50 value of >500 nM for this same competitive binding, whereas peptides containing sequences present in positions 1–14 and 1–24 of the Gla domain of human fIX did not significantly compete with [125I]‐fIX for BAEC binding. We conclude that whereas a specific receptor recognition element is present within residues 1–14 of fIX, as has previously been concluded by others and by us, full expression of this epitope requires its presence within the entire Gla domain and HS for proper folding. All determinants for proper folding of fIX that lead to BAEC receptor binding appear to be present within these two domains. © Munksgaard 1996.
Molecular weight determination of the hepatic vasopressin receptor with a high‐affinity photoprobe
Authors: BARBEAU D. , BOULEY R. , ESCHER E.
Published: August 2010

We report here a study of photoaffinity labeling of the V1a‐vasopressin receptor with high‐affinity, V1‐specific radioiodinated antagonist ligands: one containing an azidophenylalanine residue ([β, β‐dimethyl‐β‐mercaptopropionyl1,p‐azido‐Phe2, Val4, Lys8,d‐Tyr9] vasopressin), two others containing nitrophenyl‐alanine, and one, highly similar but without a photosensitive function, as control. All analogues competed in the dark for the same binding site with vasopressin. Long‐wavelength UV irradiation of rat liver membranes incubated in presence of the radio‐iodinated azido photolabel produced a specifically labeled protein band at 53 kDa in SDS‐PAGE. Identical experiments with the nitrophenylalanyl peptides produced only non‐specific labeling and control experiments with the non‐photosensitive analogue produced no labeling at all. Chemical cross linking of3H‐VP to the same membrane preparation produced a result identical to that of the azido photolabel, confirming the receptor nature of the labeled protein. Deglycosylation of the labeled receptor with endoglycosidase F reduced the observed molecular weight of 53 kDa to 43 kDa. The molecular parameters reported herein of the presumed hepatic vasopressin receptor confirm the values deduced from the molecular cloning of the rat V1areceptor. © Munksgaard 1996.
Cyclic morphiceptin analogs: cyclization studies and opioid activitiesin vitro
Authors: VOGEL D. , SCHMIDT R. , HARTUNG K. , DEMUTH H.‐U. , CHUNG N.N. , SCHILLER P.W.
Published: August 2010

Attempts were undertaken to develop cyclic β‐casomorphin‐5 analogs with improved opioid activity profiles by deletion of the glycine residue in position 5, leading to analogs structurally related to the opioid peptide morphiceptin (H‐Tyr‐Pro‐Phe‐Pro‐NH2). The tetrapeptide sequence Boc‐Tyr(tBu)‐D‐Xaa‐Phe‐Yaa‐OH (Xaa = Lys, Orn, A2bu; Yaa = Pro in L‐ or D‐configuration) was used to study the influence of ring size and chirality on the yield of cyclization between the ω‐amino group of Xaa and theC‐terminal carboxyl group. In all cases the cyclization reaction was performed under identical experimental conditions to allow a direct comparison with regard to yield and homogeneity. The reaction products were purified by crystallization and liquid chromatography, and were characterized by HPLC, TLC, electrospray mass spectrometry and1H‐NMR spectroscopy. In none of the reactions performed with the cyclization precursors containing proline in the L‐configuration could a cyclic monomer be detected, and the cyclodimer (7–9) was the exclusive product in each case. Cyclodimerization was also the favored reaction in the attempted formation of the 11‐membered ring of the cyclic [D‐A2bu2, D‐Pro4]‐morphiceptin analog12, since only traces of the monomer were found. In the case of both the [D‐Lys2, D‐Pro4]‐analog10and the [D‐Orn2, D‐Pro4]‐ analog11, the cyclomonomer/cyclodimer ratio was about 80: 20. The cyclic monomers10and11showed high opioid activity in the μ‐receptor‐representative guinea pig ileum assay (IC50= 2–5 nM) and in the δ‐receptor representative mouse vas deferens assay (IC50= 50–60 nM), whereas the potency of the cyclodimers was 2–3 orders of magnitude lower in bothin vitrobioassays.
NMR solution structure of the receptor binding domain of Pseudomonas aeruginosa pilin strain P1. Identification of a β‐turn
Authors: CAMPBELL A. PATRICIA , SHETH HASMUKH , HODGES ROBERT S. , SYKES BRIAN D.
Published: August 2010

The solution structure of the peptide antigen from the receptor binding domain of Pseudomonas aeruginosa strain P1 has been determined using two‐dimensional 1H NMR techniques. Ensembles of solution conformations for the trans form of this 23‐residue disulfide bridged peptide have been generated using a simulated annealing procedure in conjunction with distance and torsion angle restraints derived from NMR data. Comparison of the NMR‐derived solution structures of the P1 peptide with those previously determined for the 17‐residue PAK, PAO and KB7 strain peptides [Mclnnes, C., et al. (1993) Biochemistry32, 13432–13440; Campbell, A.P., et al. (1995) Biochemistry34, 16255–16268] reveals the common structural motif of a β‐turn, which may be the necessary structural requirement for recognition of a common cell surface receptor and a common cross‐reactive antibody to which all four strains bind. The importance of this conserved β‐turn in the PAK, PAO, KB7 and P1 peptides is discussed with regard to the design of a synthetic peptide vaccine effective against multiple strains of Pseudomonas aeruginosa infections.