bluuebunny/crossref_metadata_2025 huggingface dataset:
Location of the three disulfide bonds in an antimicrobial peptide from Amaranthus caudatus using mass spectrometry
Authors: BOUYOUSSFI MOUNIR EL , LAUS GEORGES , VERHEYDEN PATRICIA , WYNS LODE , TOURWE DIRK , VAN BINST GEORGES
Published: August 2010

The disulfide bridge pairing of Ac‐AMP2, a 30‐residue peptide isolated from the seeds of Amaranthus caudatus, is determined using a fast method involving enzymatic fragmentation followed by identification of the fragments with FAB mass spectrometry. The results confirm the location of the three disulfide bridges as previously established by NMR spectroscopy and molecular modelling. © Munksgaard 1997.
Stability of protease inhibitors based on the Bowman‐Birk reactive site loop to hydrolysis by proteases
Authors: GARIANI TALAL , LEATHERBARROW ROBIN J.
Published: August 2010

Bowman‐Birk proteinase inhibitor proteins contain two inhibitory regions, each of which is encapsulated within nine‐residue disulfide‐linked loops. It is known that short cyclic peptides that retain the nine‐ residue disulfide‐bridged motif have inhibitory activity, and can be used as models of the natural inhibitor protein. Two factors are important in determining the effectiveness of such inhibitor peptides: the value of the inhibition constant, and rate at which the inhibitor peptide is hydrolyzed by the proteinase. In this paper we report a study of the inhibitory properties and stability towards proteolytic hydrolysis of a family of synthetic peptides derived from the trypsin reactive site loop of the Bowman‐Birk inhibitors. The addition of a single amino acid residue to each end of the nine‐residue disulfide‐linked loop is found to reduce the rate at which the peptide is hydrolyzed. In addition, changing the P2’residue from Asn Ile gives inhibitors with considerably enhanced stability to proteolysis, as well as reduced values of K. The implications of these factors for the design of inhibitors based on this loop motif is discussed. © Munksgaard 1997.
Inhibition of homotypic adhesion of T‐cells: secondary structure of an ICAM‐1‐derived cyclic peptide
Authors: JOIS D.S. SEETHARAMA , PAL DHANANJAY , TIBBETTS SCOTT A. , CHAN MARCIA A. , BENEDICT STEPHEN H , SIAHAAN TERUNA J.
Published: August 2010

The objective of this study was to elucidate the solution conformation of cyclo‐(1, 12) Pen1‐Pro2‐Ser3‐Lys4‐Val5‐Ile6‐Leu7‐Pro8‐Arg9‐Gly10‐Gly11‐Cys12 (1) derived from the intercellular adhesion molecule‐1 (ICAM‐1). Cyclic peptide 1 inhibits homotypic adhesion of T‐cells (Molt‐3) mediated by ICAM‐1 and the leukocyte function‐associated antigen‐1 (LFA‐1) on the surface of T‐cells. Cyclic peptide 1 is more potent than is the linear peptide Pen1‐Pro2‐Ser3‐Lys4‐Val5‐Ile6‐Leu7‐Pro8‐Arg9‐Gly10‐Gly11‐Cys12 (2) in inhibiting homotypic adhesion. The difference in biological activity of peptides 1 and 2 may be due to the more stable conformation of cyclic peptide 1 compared to linear peptide 2 or because cyclization prevents the peptide from adopting non‐productive conformation. Therefore, conformational studies of cyclic peptide 1 will give a better understanding of its biological active conformation. The conformational studies of cyclic peptide 1 were done by NMR, CD and molecular dynamics simulations. NMR studies indicated that the major conformation of cyclic peptide 1 contained trans‐configuration at both X‐Pro peptide bonds. Type I β‐turns at Lys4‐Val5‐Ile6‐Leu7 and Leu7‐Pro8‐Arg9‐Gly10 were found in cyclic peptide 1. The C‐ and N‐terminal regions of this peptide were stabilized by antiparallel β‐sheet‐like structure with the presence of intramolecular hydrogen bonds. The overall structure of this peptide exposed the hydrophobic side chains on one face of the molecule and the hydrophilic side chains on the other.
Characterization of a new family of toxin‐like peptides from the venom of the scorpion Leiurus quinquestriatus hebraeus‐NMR structure of leiuropeptide II
Authors: BUISINE ERIC , WIERUSZESKI JEAN‐MICHEL , LIPPENS GUY , WOUTERS DANIÈLE , TARTAR ANDRÉ , SAUTIERE PIERRE
Published: August 2010

To extend our knowledge about the structural features of short scorpion toxins, the ion‐exchange fractions obtained from Leiurus quinquestriatus hebraeus venom were investigated by plasma desorption mass spectrometry in order to select low molecular mass polypeptides. Three toxin‐like peptides with molecular mass close to 3 kDa, named leiuropeptides I, II and III, were purified and found devoid of any significant toxicity against mammals and insects. Their amino acid sequences revealed a cysteine pattern analogous to that of short‐chain scorpion toxins. The solution structure of leiuropeptide II was determined by 2D H‐NMR spectroscopy and indicated the presence of a helix accomodating a proline, connected to a two‐stranded β‐sheet by three disulfide bonds. The overall fold of leiuropeptide II is found to be similar to that of leiurotoxin I, a 31‐residue toxin present in the same scorpion venom which acts on K+ channels. In order to rationalize the absence of toxicity, the electrostatic potential of leiuropeptide II was compared to that of leiurotoxin I. The peptide is characterized by a large negative zone around Glu4, Asp5 and Asp8 residues, beginning in the neighbourhood of the β‐turn and extending along the helix. In the same area, leiurotoxin I exhibits a positive surface, around Arg6 and Argl3 basic residues, which are essential for its receptor affinity. © Munksgaard 1997.
Membrane interaction of synthetic peptides related to the putative fusogenic region of PH‐30α, a protein in sperm‐egg fusion
Authors: NIIDOME TAKURO , KIMURA MIYUKI , CHIBA TOMOMI , OHMORI NAOYA , MIHARA HISAKAZU , AOYAGI HARUHIKO
Published: August 2010

In order to investigate the relationship between structure and function of a putative fusogenic region of PH‐30a, a protein active in sperm‐egg fusion, two peptides, SFP22 and SFP23, whose sequences correspond to the residues 90‐111 and 89‐111 of PH‐30α, respectively, were chemically synthesized. An analog of SFP23, SFP23AA, which has an Ala‐Ala sequence instead of the Pro‐Pro sequence in SFP23, was also prepared. The CD study indicated that SFP22 and SFP23 mainly took a β‐structure in the presence of DPPC and DPPC/DPPG (3/1) vesicles, while SFP23AA showed an α‐helical pattern though the a‐helical content calculated was low (25–30%). α‐Helical CD curve was observed for these peptides in trifluoroethanol. The membrane‐perturbing activity of SFP22 and SFP23 was weaker than that of SFP23AA. On the other hand, the membrane‐fusogenic activity of SFP22 and SFP23 to acidic phospholipid bilayers was much stronger than that of SFP23AA. All the peptides caused very weak cell lysis. These results are consistent with the reported speculation [Blobel, C. P. et al. (1992), Nature (London) 356, 248–252 that residues 90–111 of PH‐30α may be the fusogenic region and suggest that the Pro‐Pro sequence is one of the important factors for holding the active secondary structure of the fusogenic region of PH‐30α in membranes.
Hydrogen bonding in peptide helices Analysis of two independent helices in the crystal structure of a peptide Boc‐Val‐Ala‐Leu‐Aib‐Val‐Ala‐Phe‐OMe
Authors: DATTA SAUMEN , SHAMALA N. , BANERJEE ARINDAM , BALARAM P.
Published: August 2010

The crystal structure determination of the heptapeptide Boc‐Val‐Ala‐Leu‐Aib‐Val‐Ala‐Phe‐OMe reveals two peptide helices in the asymmetric unit. Crystal parameters are: space group P21, a =10.356(2) Å, b = 19.488(5) Å, c=23.756(6) Å, β= 102.25(2)°, V=4685.4Å3, Z=4 and R = 5.7% for 7615 reflections [I>3σ(I)].Both molecules adopt largely α‐helical conformations with variations at the C‐terminus. Helix type is determined by analysing both 4 → 1 and 5 → 1 hydrogen‐bond interactions and comparison with the results of analysis of protein structures. The presence of two 4 → 1 hydrogen‐bond interactions, besides four 5→1 interactions in both the conformations provides an opportunity to characterize bifurcated hydrogen bonds at high resolution. Comparison of the two helical conformations with related peptide structures suggests that distortions at the C‐terminus are more facile than at the N‐terminus. © Munksgaard 1997.
Antimicrobial and hemolytic activities of crabrolin, a 13‐residue peptide from the venom of the European hornet, Vespa crabro, and its analogs
Authors: KRISHNAKUMARI V. , NAGARAJ R.
Published: August 2010

The venom of insects like bee, hornet and wasp contain peptides that exhibit potent biological activities. Many of these peptides are composed of 13‐26 residues and are thus accessible through chemical synthesis as well as amenable to studies directed toward structure‐function correlations. In this report, we describe antibacterial and hemolytic activities of crabrolin: FLPLILRKIVTAL‐NH2, a 13‐residue peptide present in the venom of the hornet Vespa crabro and related peptides. The analogs were chosen so that the role of proline and positively charged amino acids in modulating biological activities could be evaluated. Our results indicate that, although helical conformation is necessary for hemolytic activity, it is not a prerequisite for antibacterial activity. Appropriately positioned, charged and hydrophobic residues and overall hydrophobicity appear to determine antibacterial activity. The discovery of a large number of host‐defense peptides in a variety of species in recent years offers a large repertoire of molecules that can be “engineered” based on biophysical principles to yield molecules with specific activities.
Structural studies by H NMR of a prototypic α‐helical peptide (LYQELQKLTQTLK) and homologs in trifluoroethanol/water and on sodium dodecyl sulfate micelles
Authors: YOUNG JOHN K. , MARí FRANK , XU MINZHEN , HUMPHREYS ROBERT E. , CLEMENTE NIKKI M. , STATTEL JAMES M. , NELSON DONALD J. , GAMBINO JOSEPH , WRIGHT GEORGE E.
Published: August 2010

The H NMR‐determined structure and dynamics of a synthetic, amphiphilic α‐helical peptide, PH‐1.0 (LYQELQKLTQTLK), and several homologs were compared in 50% trifluoroethanol‐d2 (TFE‐d2)/H2O and in sodium dodecyW25 sulfate (SDS‐d25) micelles. The peptides were designed to test the influence on secondary structure of placement of favored and disfavored residues relative to a “longitudinal, hydrophobic strip‐of‐helix” defined by the repeating leucines. PH‐1.0 was highly ordered as an α‐helix in 50% TFE‐d2/H2O and in SDS‐d25 micelles. Homologs PH‐1.1, in which LI was replaced by T, and PH‐1.4, in which LI 2 was replaced by T, were found to be partially helical in both media. Calculated structures in SDS‐d25 revealed that the helix of PH‐1.1 was slightly disordered at the N‐terminus, but that of PH‐1.4 was completely disordered at the C‐terminus. Examination of distributions of hydrophobic residues in protein structures revealed that, when ♦= LIVFM and ⋄= nonLIVFM, the pattern ⋄♦♦⋄⋄ is favored and ⋄♦⋄♦⋄ is disfavored in α‐helices. Several analogs of PH‐1.0 incorporating these patterns were studied. Peptide PH‐1.12 (LYQELQKLLQTLK) retained a‐helical structure in both 50% TFE‐d2/H2O and in SDS‐d25 micelles. However, although PH‐1.13 (LYQELQKLLQTLK) was fully helical in 50% TFE, it was helical only through residue 6 in SDS micelles. Two homologs containing an additional loop of the helix and repeats of favored (PH‐5.0, NYLQTLLETLKTLLQK) or suppressed LL patterns (PH‐5.11, NYLQTLELTLKLTLQK) gave similar results, i.e. the latter peptide was helical only through residue 6 in SDS micelles. The degree of local order in these SDS micelle‐adsorbed peptides correlates to placement of hydrophobic residues in motifs which are favored or disfavored in proteins in general and in α‐helices specifically. © Munksgaard 1997.
β‐Turn induction by C60‐based fulleroproline: synthesis and conformational characterization of Fpr/Pro small peptides
Authors: BIANCO A. , BERTOLINI T. , CRISMA M. , VALLE G. , TONIOLO C. , MAGGINI M. , SCORRANO G. , PRATO M.
Published: August 2010

We have recently described the preparation of Fpr (C60‐based fulleroproline). In this paper the synthesis and a conformational characterization of heterochiral di‐and tripeptides containing this new α‐amino acid are reported. A folded structure, induced by the‐l‐Fpr‐d‐Ala‐sequence in chloroform solution and detected by Fourier transform infrared absorption and H nuclear magnetic resonance, has been compared with the known propensity of the cognate‐l‐Pro‐d‐Ala‐sequence to adopt a βII‐turn conformation, which has also been confirmed in this work. The βII‐turn structure is retained in the crystal state by the Pro‐peptides, as shown by the X‐ray diffraction structures of Ibu‐l‐Pro‐d‐Ala‐NHtBu and Z‐l‐Pro‐d‐Ala‐l‐Ala‐OtBu. © Munksgaard 1997.
Para‐Substituted phenylalanine‐4 analogues of [l‐Ala3]. DPDPE: highly selective δ opioid receptor ligands
Authors: HAASETH RONALD C. , ZALEWSKA TERESA , DAVIS PEG , YAMAMURA HENRY I. , PORRECA FRANK , HRUBY VICTOR J.
Published: August 2010

Several para‐substituted Phe4analogues of the δ1‐selective antagonist [l‐Ala3]. DPDPE (DPADPE) were prepared and evaluated for their brain‐binding andin vitropharmacological effects. Unlike thep‐haloPhe4analogues of DPDPE and the deltorphins, similar analogues of DPADPE with electron‐withdrawing groups substituted at the para‐position of the Phe4aromatic ring did not all have increased potency and selectivity for δ opioid receptors, but all retained high potency and selectivity for δ opioid receptors greater than DPDPE. © Munksgaard 1997.
Antiproliferative and antitumor activities of D‐reverse peptides derived from the second type‐1 repeat of thrombospondin‐1
Authors: GUO NENG‐HUA , KRUTZSCH HENRY C. , INMAN JOHN K. , SHANNON CAITLIN S. , ROBERTS DAVID D.
Published: August 2010

The extracellular matrix glycoprotein thrombospondin‐1 (TSP1) inhibits angiogenesis, endothelial cell growth, motility and adhesion. Peptides from the type I repeats of TSPl mimic the adhesive and growth inhibitory activities of the intact protein and specifically interact with heparin and transforming growth factor‐β (TGFβ). To define the structural basis for the antiangiogenic activities of these peptides, we prepared analogs of the TSPl peptide KRFKQDGGWSHWSPWSSC. l‐forward, l‐reverse, and d‐reverse (retro‐inverso) analogs displayed identical activities for binding to heparin. demonstrating a lack of stereospecificity for heparin binding. The l‐reverse and d‐reverse peptides, however, had somewhat decreased abilities to activate latent TGFβ. Conjugation of the forward peptides through a C‐terminal thioether and the reverse peptides through an N‐terminal thioether to polysucrose abolished the adhesive activity of the peptides and enhanced their antiproliferative activities for endothelial and breast carcinoma cells stimulated by fibroblast growth factor‐2. Their antiproliferative activities were independent of latent TGFβ activation, because substitution of an Ala residue for the essential Phe residue in the TSPl type‐1 repeat peptide increased their potency for inhibiting TSPl binding to heparin and for inhibiting endothelial cell proliferation. Although the conjugated peptides were inactive in vivo, an unconjugated retro‐inverso analog of the native TSP peptide inhibited breast tumor growth in a mouse xenograft model. Thus, these TSP‐derived peptide analogs antagonize endothelial growth through their heparin‐binding activity rather than through activation of latent TGFβ or increasing cell adhesion. These stable analogs may therefore be useful as therapeutic inhibitors of angiogenesis stimulated by fibroblast growth factor‐2. © Munksgaard 1997.
Synthesis and biological activities of head‐to‐tail cyclic bradykinin analogues of varying ring size
Authors: GOBBO MARINA , BIONDI LAURA , CAVAGGION FRANCO , FILIRA FERNANDO , PIEK TOM , MANTEL PIET , ROCCHI RANIERO
Published: August 2010

Syntheses of cyclic kinin analogues with different backbone atom numbers are described. Cyclization, by either the O‐benzotriazolyl‐N, N, N', N'‐tetramethyluronium tetrafluoroborate/1‐hydroxybenzotriazole/diisopropylethyl amine (TBTU‐HOBt‐DIPEA) or the diphenylphosphoryl azide (DPPA) procedure of linear peptides prepared by the solid‐phase method based on the g‐fluorenyl methyloxycarbonyl chemistry, was used for preparing cyclo‐Gly‐Ile‐Ile‐Gly‐bradykinin, cyclo‐Lys‐kallidin (cyclo‐Lys‐Lys‐bradykinin) and cyclo‐des Arg‐bradykinin. Peptides were characterized by amino acid analysis, optical rotation, analytical high‐performance liquid chromatography and matrix‐assisted laser desorption ionization‐time flight mass spectrometry. Pharmacological experiments showed that cyclo‐Gly‐Ile‐Ile‐Gly‐bradykinin (39 backbone atoms) and cyclo‐Lys‐bradykinin (30 backbone atoms) are about equipotent, when tested on the relaxation of the isolated rat duodenum preparation. The potency of cyclo‐des Arg‐bradykinin is at least three orders of magnitude lower. The potency of cyclo‐Lys‐Lys‐bradykinin (33 backbone atoms) is one tenth the activity of bradykinin but about 10 times higher than the potency of the above‐mentioned cyclokinins and makes the latter analogue the most potent end‐to‐end cyclic analogue known currently. The present results, in agreement with data from earlier reports, seem to indicate that the enhancement of the number of backbone atoms in the cyclic kinins first increases and subsequently decreases the potency, whereas a reduction in the atom number from 27 to 24 causes a dramatic decrease in potency.
The nature of trypsin‐pancreatic trypsin inhibitor binding: free energy calculation of Tyr39→ Phe39 mutation in trypsin
Authors: MELO A. , RAMOS M. J.
Published: August 2010

The main goal of this work is the detailed study of the binding interactions in the trypsin‐pancreatic trypsin inhibitor (PTI) complex and here, we present how meaningful the Tyr39‐Ile19 interaction is to the stability of that particular complex using free energy methods. This knowledge should be very important in the design of new inhibitors for trypsin and enzymes homologous to it. In particular, it could help to decide whether it is possible to produce selective inhibitors for these enzymes by appropriate mutations of residues in the contact region of PTI.
Structure and supramolecular packing features of the dipeptide Arg‐Val acetate
Authors: RECACHA ROSARIO , VERDAGUER NÚRIA , SUBIRANA JUAN A.
Published: August 2010

The title compound crystallizes in the zwitterionic form. The crystal forms a supramolecular structure with the peptide molecules organized in head‐to‐tail columns in the b direction. The arginine side‐chains and acetate ions interact with neighbor peptides in the c direction. Infinite hydrophobic columns are present in the a direction; they involve the valine side‐chains, the acetate methyl groups and the methylene groups of the arginine side‐chains. This three‐dimensional organization is similar to that found in Lys‐Val hydrochloride. © Munksgaard 1997.
Nuclear magnetic resonance spectroscopy of mussel adhesive protein repeating peptide segment
Authors: OLIVIERI MARION P. , WOLLMAN ROBERT M. , ALDERFER JAMES L.
Published: August 2010

Mussel adhesive protein (MAP) is the adhesive agent used by the common blue sea mussel (Mytilus edulis) to attach the animal to various underwater surfaces. It is generally composed of 75 to 85 repeating decameric units with the reported primary sequence NH2‐Ala(1)‐Lys(2)‐Pro(3)‐Ser(4)‐Tyr(5)‐Hyp(6)‐Hyp(7)‐Thr(8)‐DOPA(9)‐Lys(10)‐COOH. This study examines this peptide's solution‐state conformation using proton nuclear magnetic resonance (NMR) spectroscopy. NMR and molecular modeling of the decamer before and after molecular dynamics calculations in water suggests a conformation that retains an overall bent helix. © Munksgaard 1997.
Structural characterization of the molten globule and native states of ovalbumin: A H NMR study
Authors: SOGAMI MASARU , ERA SEIICHI , KOSEKI TAIHEI , NAGAI NAOKI
Published: August 2010

Molecular characteristics of ovalbumin (OVA) in the acidic (pD 3.08, the E‐form) and neutral [pD 7.29, the N‐form (native form)]. regions were studied by measuring effective radii, H NMR spectra, spin‐echo H NMR spectra and cross‐relaxation times (TIS) from irradiated to observed protein protons which are particularly sensitive for detection of the mobile segments and/or structural looseness in proteins. H NMR spectra did not show significant differences between the N‐ and E‐forms except for the spectral lines in the CH3, °CH2 and aromatic regions. Effective radii and TIS values for main‐ and side‐chains showed 1.08 and 1.5‐ to 2.0‐fold increases on going from the N‐ to E‐forms, respectively. The elongation of TIS values might indicate the appearance of the fluctuating tertiary structure in the E‐form. Molecular characteristics of the E‐form, inferred from reported far ultraviolet‐circular dichroism (UV‐CD) spectra in the peptide region, near UV‐CD spectra in the aromatic region [Koseki et al. (1988) J. Biochem.103, 425‐130]., effective radii and especially elongation of TIS values might indicate that the E‐form could be in the molten globule state. The onset of denaturation of OVA using I is measurements was also studied.
Epitopic characterization of the human wild‐type and mutant ras proteins using membrane‐bound peptides
Authors: WANG ZHENGXIN , CARNEY WALTER P. , LAURSEN RICHARD A.
Published: August 2010

Overlapping octapeptides encompassing the entire sequences of the human oncogene products Ha‐ras, K‐ras and N‐ras protein were synthesized as spots on polypropylene membrane sheets. The binding of anti‐row protein monoclonal antibodies (mAbs) to the membrane‐bound peptides was assessed using an enzyme‐linked immunosorbent assay. Epitopes of 10 of 18 mAbs to the human ras proteins were mapped and identified by this procedure. The epitopes of nine of the mAbs are within residues 28‐39 in the constant domain common to the three ras proteins, whereas the epitope of the tenth (mAb 21) spans residues 136‐144 in Ha‐ras. The minimal lengths of epitopes of all ten of the mAbs were further precisely mapped using peptides of varying length, and the tolerance for mAb binding of mutated epitopes was determined by systematically replacing each residue in the epitope with each of the 20 common amino acids. The results show that most of these mAbs have essentially the same binding specificity, namely for the sequence YDPT (residues 32–35) or for slightly longer sequences containing these residues. This site is in the switch 1 region (residues 32‐38) in the ras effector loop, indicating that some of the same residues important for the interaction of ras with other proteins (GTPase‐activating protein, neurofibromin or raf) are highly antigenic. In addition, we investigated epitopes and specificity of five mAbs against the activated human ras proteins by the same procedure. The information gained from this study should be useful both for study of the complicated functions of ras proteins and for clinical detection of ras oncogenes in human tumor cells.
Synthesis and biological activity of potent, low molecular weight renin inhibitors*
Authors: SUEIRAS‐DIAZ JAVIER , JONES D. MICHAEL , SZELKE MICHAEL , DEINUM JOHANNA , SVENSSON LENNART , WESTERLUND CHRISTER , SOHTELL MORGAN
Published: August 2010

A series of renin inhibitors containing the dipeptide transition state mimics (2R, 4S, 5S)‐5‐amino‐4‐hydroxy‐2‐methyl‐6‐cyclohexyl hexanoic acid (Cha‐Ψ[CH(OH)CH2]Ala) and (2R, 45, 5S)‐5‐amino‐4‐hydroxy‐2‐isopropyl‐6‐cyclohexyl hexanoic acid (Cha‐Ψ[CH(OH)CH2]Val) were prepared. A structure‐activity study, using pseudopeptide (Boc‐Phe‐His‐Leu‐Ψ[CH(OH)CH2]Val‐Ile‐His‐OH) as our lead structure, led to a new series of inhibitors, which correspond to tripeptides and contain no natural amino acids. For example, R, S‐Bpma‐Ape‐Cha‐Ψ[CH(OH)CH2]Ala‐NH2 (IC50= 1.26 nM against human plasma renin at pH 6.0; molecular weight = 564) has only two thirds of the molecular weight but twice the potency of our original lead. This new class of low molecular weight renin inhibitor displays excellent specificity toward human renin versus the related aspartic proteinase pepsin and angiotensin‐1‐converting enzyme. Examples are given of selected inhibitors showing encouraging evidence for intestinal absorption after intracolonic and oral administration in male Sprague‐Dawley rats.
Synthesis and biological activities of fluorescent acridine‐containing HIV‐1 nucleocapsid proteins for investigation of nucleic acid‐NCp7 interactions
Authors: DONG C.Z. , DE ROCQUIGNY H. , RÉMY E. , MELLAC S. , FOURNIÉ‐ZALUSKI M.C. , ROQUES B.P.
Published: August 2010

Specific interactions between the 72‐amino acid nucleocapsid protein NCp7 of the human immunodeficiency virus, type 1 and the genomic RNA are essential for virus replication. Studies on the mechanism of action of NCp7 require a direct visualization of its complexes with nucleic acids and the determination of binding affinities. To facilitate these investigations, fluorescent NCp7 derivatives were developed by introduction in the NCp7 sequence of a non‐natural amino acid, (S)‐β‐(9‐acridinyl)alanine (Aca) obtained by a chiral synthetic method. Three fluorescent NCp7 derivatives were obtained by introducing this amino acid at different positions. As shown by NMR, the three‐dimensional structure of NCp7 is not altered by introduction of Aca. The fluorescent peptides were found to be as potent as their precursors in interacting with nucleic acids and in promoting HIV‐1 genomic RNA dimerization. Moreover, because of their fluorescent properties, these NCp7s can be used at submicromolar concentrations to directly visualize and quantify protein‐nucleic acid interactions in solution or after gel electrophoresis. This could facilitate the development of new antiviral agents aimed at inhibiting the functions of NCp7 and studies on the intracellular traffic of NCp7 within the preintegration complex.
Granulocyte‐colony stimulating factor maintains a thermally stable, compact, partially folded structure at pH 2
Authors: KOLVENBACH CARL G. , NARHI LINDA O. , PHILO JOHN S. , LI TIANSHENG , ZHANG MEI , ARAKAWA TSUTOMU
Published: August 2010

At acidic pH many proteins exist in a partially unfolded form, called the “A” state. This is defined as a flexible, expanded structure with well‐defined, usually native‐like secondary structure, but no unique tertiary structure, and showing no cooperativity during thermal‐induced denaturation. Granulocyte‐colony stimulating factor (G‐CSF), a four‐helix bundle cytokine, maintains both thermal stability and tertiary structure at pH 2.O. We therefore examined the conformation and thermal unfolding of G‐CSF at pH 2.0, 4.0 and 7.0 using circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR). The secondary structure of the molecule remains highly helical as the pH is lowered from 7.0 to 2.0. The tertiary structure of the protein is slightly different at each pH value, but even at pH 2.0 G‐CSF maintains a regular three‐dimensional structure. The structure is hydrodynamically compact at these different pH values, with no increase in Stake's radius even at pH 2.0.The thermal‐induced denaturation of G‐CSF was determined by monitoring changes in the CD or FTIR spectra. At pH 2.0 the temperature at which thermal‐induced denaturation begins is higher than it is at pH 4.0 or 7.0, the thermal unfolding transition remains cooperative and some α‐helical structure persists even at 86°C. At pH 4.0 and 7.0, secondary and tertiary structures disappear simultaneously during thermal denaturation, whereas at pH 2.0 small changes in the far‐UV CD region begin to occur first, followed by the simultaneous cooperative loss of tertiary structure and much of the remaining secondary structure. The structure of G‐CSF at pH 2.0 is thus revealed as compact, with a unique, three‐dimensional structure, highly helical secondary structure, and most importantly, a cooperative thermal unfolding transition. G‐CSF at acid pH thus does not adopt the “A” state.
Direct synthesis and characterisation of multi‐dendritic peptides for use as immunogens
Authors: KEAH HOOI HONG , KECORIUS ELAINE , HEARN MILTON T.W.
Published: August 2010

The direct synthesis and subsequent rapid characterisation of multiple antigen peptides (MAPs) for use as immunogens has presented difficulties, partly because of the formation of incomplete or truncated peptide sequences during the synthetic procedure. Therefore, many researchers have resorted to ligation procedures for the synthesis of MAP constructs. This article describes a method to improve the yield of MAP constructs by direct synthesis methods, as well as a general procedure that enables easier characterisation of the synthetic products. In particular, during the synthesis of MAP constructs, a capping procedure was introduced after each amino acid coupling step, thus improving significantly the yield of me desired multi‐dendritic peptidic immunogens. Through the use of this capping procedure, problems arising from the incomplete amino acid residue coupling at the point of synthesis were minimised, and any deletion peptides which formed could be eliminated more readily during the subsequent purification procedures. In addition, previous difficulties in purification and characterisation of MAP construct by, e.g. electrospray mass spectroscopy (ES‐MS), often led to the multi‐dendritic peptidic immunogens being used without full characterisation after dialysis and recovery of the product(s). This article describes an enzymatic (tryptic) digestion method with the MAP construct, followed by characterisation of the enzymatic digest by reversed phase high‐performance liquid chromatography‐ES‐MS. With this method, fragments of the MAP construct cleaved at specific amino acid residue sites (e.g. lysine or arginine) within the sequence of the parent peptide can be readily determined and the kinetics of the digestion easily followed. This enzymatic digestion procedure thus provides a facile approach to confirm that all of the multi‐dendritic arms of the purified MAP construct have been equivalently elongated during the peptide synthesis and that consequently the purified construct structure contains the correct peptide sequence.
Inactivation and conformational changes of yeast invertase during unfolding in urea and guanidinium chloride solutions
Authors: LI SEN , YANG HAI‐PENG , ZHOU HAI‐MENG
Published: August 2010

Yeast invertase exists in two different forms. The cytoplasmic enzyme is non‐glycosylated, whereas the external invertase contains approximately 50% carbohydrate of the high mannose type. In this paper, the inactivation and the conformational changes of the yeast external invertase are analyzed for unfolding in urea and guanidinium chloride. The results show that much lower concentrations of denaturants are required to bring about inactivation than are required to produce significant conformational changes of the yeast external invertase. The results suggest that the active sites of the external invertase containing carbohydrate residues may display more conformational flexibility than the enzyme molecules as a whole.
Comparative conformational studies on cyclic hexapeptides corresponding to message sequence His‐Phe‐Arg‐Trp of α‐Melanotropin by NMR
Authors: PRACHAND MAMATA S. , DHINGRA M. M. , SARAN ANIL , COUTINHO EVANS , BODI J. , SÜLI‐VARGHA H. , MEDZIHARDSZKY K.
Published: August 2010

Solution conformation of cyclo(Gly1‐His2‐Phe3‐Arg4‐Trp5‐Gly6) and its d‐Phe analog corresponding to the message sequence [Gly‐α‐MSH5‐10] of α‐MSH has been studied by 1D and 2D proton magnetic resonance spectroscopy in dimethyl sulfoxide (DMSO)‐d6 solution and in a DMSO‐d6/H2O cryoprotective mixture. The NMR data for both the analogs in solution at 300 K cannot be interpreted based on a single ordered conformation, as evidenced by the broadening of only ‐NH resonances as well as the temperature coefficients of the amide protons. An analysis of the nuclear Overhauser effect (NOE) cross‐peaks in conjunction with temperature coefficient data indicates an equilibrium of multiple conformers with a substantial population of particular conformational states at least in the d‐analog. The molecular dynamics simulations without and with NOE constraints also reveal numerous low‐energy conformers with two γ‐turns, a γ‐turn and a β‐turn, two β‐turns, etc. for both the analogs. The observed NMR spectra can be rationalized by a dynamic equilibrium of conformers characterized by a γ‐bend at Gly6, two γ‐bends at Phe3 and Gly6 and a conformer with a single β‐turn and a γ‐bend for the l‐Phe analog. On the other hand, a conformation with two fused β‐turns around the two tetrads His2‐d‐Phe3‐Arg4‐Trp5 and Trp5‐Gly6‐Gly1‐His2 dominates the equilibrium mixture for the d‐Phe analog. For the d‐Phe analog, the experimentally observed average conformation is corroborated by molecular dynamics simulations as well as by studies in cryoprotective solvent.
Synthesis and conformational analysis of two 2‐oxopiperazine‐containing tetrapeptide analogues
Authors: POHLMANN ADRIANA , GUILLAUME DOMINIQUE , QUIRION JEAN‐CHARLES , HUSSON HENRI‐PHILIPPE
Published: August 2010

One unsubstituted and one stereoselectively prepared 3‐(S)‐substituted‐2‐oxopiperazine have been used as dipeptide templates to generate tetrapeptide analogues. NMR analysis shows that these tetrapeptide analogues present an inverse γ‐turn conformation in chloroform.
New antibiotic caerin 1 peptides from the skin secretion of the Australian tree frog Litoria chloris. Comparison of the activities of the caerin 1 peptides from the genus Litoria
Authors: STEINBORNER SIMON T. , CURRIE GRAEME J. , BOWIE JOHN H. , WALLACE JOHN C. , TYLER MICHAEL J.
Published: August 2010

The skin glands of the tree frog Litoria chloris contain a variety of peptides including four antibacterial peptides of the caerin 1 family. Two of these, caerins 1.6 and 1.7, are also present in the related species Litoria xanthomera. The other two peptides, caerins 1.8 and 1.9, are new. Their sequences are: GLFKVLGSVAKHLLPHVVPVIAEKL‐NH2 [Caerin 1.8] and GLFGVLGSIAKHVLPHVVPVIAEKL‐NH2 [Caerin 1.9]. Comparison of the skin peptide profiles of L. chloris and L. xanthomera confirms that these species are more closely related to each other than to any other species of the genus Litoria that we have studied. A comparison is made of the antibiotic activities of nine members of the caerin 1 family of peptides isolated from tree frogs of the genus Litoria.
α‐(Ac)AKRHRKV, a model of the histone H4 amino terminus, uses an unprotonated histidine in phosphate binding
Authors: VU HAI MINH , MINCH MICHAEL J.
Published: August 2010

The 1H NMR spectrum of the title peptide at pH 3.3 in 90% H2O was assigned by HOHAHA and NOESY 2D methods. Titration studies in D2O at 300 MHz indicated a histidine side‐chain pKa of 6.3. Peptide backbone NH resonances were studied in 90% H2O at 500 MHz as a function of pH and added phosphate. In acidic solution the peptide was free from conventional secondary structural elements, but near neutrality the valine amide proton resonance remained a sharp doublet, which suggests that it may form a hydrogen bond with some backbone carbonyl group. The other amide resonances broadened and showed significant saturation transfer from the water signal indicating that they exchange with solvent although not all to the same extent. Marked changes in the chemical shift of the histidine aromatic protons in the presence of phosphate and a 70‐fold increase in the 31P line width of inorganic phosphate in the presence of peptide only at pH values above the pKa (6.3) of the histidine imidazole side‐chain implied that the unprotonated imidazole group is specifically involved in phosphate binding. The peptide binds inorganic phosphate with a dissociation constant of 1.6 × 10−5 M−1 at pH 7.4.
Structure of cyclic peptides: the crystal and solution conformation of cyclo(Phe‐Phe‐Aib‐Leu‐Pro)
Authors: ZANOTTI GIANCARLO , SAVIANO MICHELE , SAVIANO GABRIELLA , TANCREDI TEODORICO , ROSSI FILOMENA , PEDONE CARLO , BENEDETTI ETTORE
Published: August 2010

A solid‐state and solution conformation analyses of the cyclopentapeptide cyclo(Phe‐Phe‐Aib‐Leu‐Pro) has been carried out by X‐ray diffraction and nuclear magnetic resonance techniques. The structure of the hexagonal crystals, grown from a methanol solution [a=b= 16.530(4) Å, c= 21.356(9) Å, space group P65, Z = 6], shows the presence of one intramolecular N‐H⋯O=C hydrogen bond with the formation of a γ‐turn (C7). The Aib3 residue, at the center of the γ‐turn, presents unexpected values of the torsion angles [φ= 70.5° and ψ= ‐73.8°], which have been observed only once before for this helicogenic residue. A cis peptide bond occurs between Leu4 and Pro5; all other peptide bonds are trans. The overall conformation for the cyclopentapeptide with one cis‐peptide bond on one side and an intramolecular γ‐turn on the opposite side results in an equatorial topology of the side‐chains of the Phe1, Phe2 and Leu4 residues. Indeed, the Cα‐Cβand Cβ‐Cγ bonds of these residues lie approximately in the mean plane of the cyclic ring system.

The structure is compared with data in the literature on cyclic pentapeptides. In addition the Pro‐Phe‐Phe moiety shows a conformation similar to that observed in other larger cyclic bioactive peptides, which indicates a reduced number of conformations for this sequence. The solution study was carried out in three different solvent systems: chloroform, acetonitrile and methanol in the temperature interval 220–300 K. In all three solvents the room temperature spectra show that the peptide is conformationally nonhomogeneous. In acetonitrile at low temperatures it is possible to reduce the conformational equilibrium to two predominant conformers which differ for the cis‐trans isomerism of the Leu4‐Pro5 peptide bond.
Azaproline as a β‐turn‐inducer residue opposed to proline
Authors: Zouikri M. , Vicherat A. , Aubry A. , Marraud M. , Boussard G.
Published: August 2010

Abstract:Azaproline (AzPro) is an analogue of proline containing a nitrogen atom in place of the CαH group. AzPro has been introduced in various model peptides, and especially in the Boc‐Ala‐AzPro‐Ala‐NHiPr tripeptide. The structural consequence of that modification has been investigated in solution by using IR and1H NMR, with reference to the cognate proline‐containing peptide. Contrary to proline, which induces β‐folding of the Pro‐Ala sequence, azaproline apparently favors βVI‐folding of the Ala‐AzPro one with high occurrence. Opening of the AzPro pyrazolidine ring to getN‐methylazaalanine fundamentally does not change the structural properties of the azatripeptide, but allows the existence of open conformers to an extent depending on the solvent.
Solution conformational analysis of sodium complexed [Gly6]. ‐ and [Gly9]. ‐antamanide analogs
Authors: AMODEO PIETRO , SAVIANO GABRIELLA , BORIN GIANFRANCO , CALDERAN ANDREA , RUZZA PAOLO , TANCREDI TEODORICO
Published: August 2010

To investigate the conformational flexibility of metal‐complexed cyclodecapeptides, we synthesized and studied two antamanide analogs, in which the phenylalanine residue in position 6 or 9 of the sequence was substituted by Gly. Previous conformational studies on antamanide suggested that these backbone regions are affected by conformational variation. The NMR conformational study showed a high degree of flexibility for the two analogs. With sodium ions, on the other hand, [Gly9]. ‐antamanide was able to form a fairly stable equimolar complex, whereas [Gly6]. ‐antamanide showed a conformational heterogeneity, with one prevailing conformer.

For the [Gly9]. ‐antamanide analog, the whole NMR data, combined with extensive theoretical calculations, were consistent with the presence of 1) two (β‐turns of type I, centered on Gly9‐Phe10 and Ala4‐Phe5, respectively; 2) a central cavity with a six‐carbonyl oxygen cage, optimal for a Na+ hexacoordination; 3) strongly H‐bonded amide protons for residues 1 and 6, both involved in the formation of the two type I β‐turns, which, however, exhibited some fluctuations during the molecular dynamics simulations. For the [Gly6]. ‐antamanide‐Na+ complex the prevailing conformer was consistent with a more open structure, with the partial solvent exposure of all the amide protons; that is, the Gly residue in position 6 increases the flexibility of this critical site more than does the Gly in position 9. These data in some way parallel the results of the cytotoxicity tests on B16‐F10 transformed cells for the two analogs: [Gly9]. ‐antamanide is cytotoxic after 48 h exposure, whereas [Gly6]. ‐antamanide is almost inactive. On the contrary, both analogs are practically inactive in vivo against phalloidin.
Identification of a sequence‐dependent reversible acylation of tosylarginine in a peptidyl‐resin reacted with isonicotinyl p‐nitrophenylcarbonate
Authors: MELNYK O. , CHAURAND P. , ROMMENS C. , DROBECQ H. , WIERUSZESKI J. M. , SPENGLER B. , GRAS‐MASSE H.
Published: August 2010

In this paper, we report on the identification of an unexpected acylation that occurred on the solid phase when a peptide containing an unprotected lysyl and a tosyl‐protected arginyl residue was treated with a large excess of isonicotinyl p‐nitrophenylcarbonate. NMR and matrix‐assisted laser desorption/ionisation ‐post‐source decay analysis of the purified peptide demonstrated the presence of one extra isonicotinyloxycarbonyl (iNoc) group located on the ω nitrogen atom of the arginine which was adjacent to the Lys(iNoc). The desired peptide was obtained by quantitative removal of the unwanted iNoc group during a brief treatment with diluted aqueous hydrazine.
A peptide inhibitor of cholesteryl ester transfer protein identified by screening a bacteriophage display library
Authors: BONIN PAUL D. , BANNOW CAROL A. , SMITH CLARK W. , FISCHER H. DAVID , ERICKSON LAURENCE A.
Published: August 2010

We screened a bacteriophage display library of random decapeptides to identify peptide inhibitors of cholesteryl ester transfer protein (CETP). After affinity selection against CETP, bacteriophage‐infected Escherichia coli were plated at clonal density and 36 random clones were isolated. Analysis of the relevant portion of the bacteriophage DNA from a group of 12 clones that had a relatively high affinity for CETP revealed that the corresponding amino acid sequences of the displayed peptides exhibited an. Xaa‐Arg‐Met‐Arg‐Tyr‐Xaa composite motif. Based on those results, decapeptides from this group were synthesized and one of them, DPI (NH2‐VTWRMWYVPA‐COOH), inhibited CETP‐catalyzed transfer of cholesteryl esters and triglycerides. Amino‐ and carboxy‐terminal truncations of DPI demonstrated that the original decapeptide could be reduced to a pentapeptide without loss of either its ability to bind to CETP or its ability to inhibit CETP‐mediated lipid transfer. That pentapeptide, NH2‐WRMWY‐COOH (WRMWY, PNU‐107368E), binds directly to CETP and its inhibition is consistent with that of a competitive inhibitor of CETP with a Ki of 164 μm. WRMWY or modified versions of this peptide may be useful in studying the interactions between CETP and plasma lipoproteins.
Synthesis of cyclic dipeptide templates, their incorporation into peptides and studies on their conformational and biological properties
Authors: ASCHE GEERT , KUNZ HORST , NAR HERBERT , KÖPPEN HERBERT , BRIEM HANS , POOK KARL‐HEINZ , SCHILLER PETER W. , CHUNG NGA N. , LEMIEUX CAROLE , ESSER FRANZ
Published: August 2010

This study investigated the diastereoselective synthesis of three dipeptide templates 1, 2 and 3, which may be regarded as conformationally restricted analogs of H‐Gly‐Xaa‐OH, in which Xaa constitutes an aromatic amino acid. Bond formation between α‐C of Gly and the aromatic moiety was achieved by proton‐catalyzed intramolecular electrophilic aromatic substitution. The absolute configuration of the dipeptide templates was determined by single‐crystal X‐ray crystallography or by nuclear Overhauser enhancement measurements. A protective group strategy was elaborated to allow their incorporation into peptide sequences by liquid phase as well as by solid‐phase peptide synthesis. The templates were used to generate an enkephalin analog 15, a modified peptidic neurokinin antagonist 20 and two dermorphin derivatives (24 and 33). Molecular dynamic simulations with 15 and 20 revealed the preference for a turn‐like motif for 15. The biological activity, as investigated by respective receptor binding and functional assays, was strongly diminished with all four derivatives, indicating that their receptor‐relevant molecular geometries lie outside the examined conformational space. © Munksgaard 1998.
Dipeptidyl aminotransferase activity and in vitroO‐glycosylation of MUCSAC mucin motif peptides by human gastric microsomal preparations
Authors: SOUDAN BENOIT , TETAERT DANIEL , HENNEBICQ SYLVIANE , BRIAND GILBERT , ZERIMECH FARID , RICHET COLETTE , DEMEYER DOMINIQUE , GAGNON JEAN , PETILLOT YVES , DEGAND PIERRE
Published: August 2010

The in vitroO‐glycosylation reaction of the MUCSAC mucin motif peptide, TTSAPTTS (in one‐letter code), was achieved with human gastric microsomal homogenates. The analyses using capillary electrophoresis online coupled with electrospray mass spectrometry and further Edman degradation of the purified products (obtained by capillary electrophoresis at preparative scale) allowed us to distinguish two components at close masses: the addition of a mass of 202 corresponded to an N‐terminal elongation of the peptide TTSAPTTS with the dipeptide (TT) and the addition of a mass of 203 corresponded to an N‐acetylgalactosamine O‐linkage. Using different peptidase inhibitors, a dipeptidyl peptidase/transferase activity was further characterized. A thiol dependence and an inhibition by H‐Gly‐PheCHN2 (specific to cathepsin C activity) were found. Moreover, besides TTSAPTTS, other MUCSAC motif peptides (GTTPSPVP, TSAPTTS) were also dipeptide donors (GT and TS, respectively) and our results suggested the involvement of a single dipeptidyl peptidase/transferase activity. Finally, this latter activity modified the in vitro GalNAc incorporation rates when using our selected MUCSAC motif peptides. Our study therefore shows that caution must be taken to prevent peptidic substrate elongation while performing in vitroO‐glycosylation with microsomal preparations as the enzyme source. In fact, the results of the N‐acetylgalactosamine incorporation rates and thus the microsomal N‐acetylgalactosamine transferase affinity can be misinterpreted if dipeptidyl peptidase/transferase activity is not inhibited by the thiol inhibitor E‐64 or the cathepsin C inhibitor H‐Gly‐PheCHN2. © Munksgaard 1998.
Solution conformations of deltorphin‐I obtained from combined use of quantitative 2D‐NMR and energy calculations: A comparison with dermenkephalin
Authors: Naim M. , Nicolas P. , Benajiba A. , Baron D.
Published: August 2010

Deltorphin‐I, Tyr‐d‐Ala‐Phe‐Asp‐Val‐Val‐Gly‐NH2and dermenkephalin, Tyr‐d‐Met‐Phe‐His‐Leu‐Met‐Asp‐NH2, two highly related opioid peptides from frog skin, display very similar N‐termini but strikingly different C‐terminal tails. Nevertheless, both peptides are highly potent at, and exquisitely selective for the δ‐opioid receptor. To identify common determinants concuring to the remarkably efficient targeting of deltorphin‐I and dermenkephalin, combined use of quantitative two‐dimensional nuclear magnetic resonance (53 dipolar interactions studied at four temperatures) and energy calculations using simulated annealing generated five groups of deltorphin‐I conformers. These groups were pooled into two families whose overall conformation could be described either by a left‐handed helix (Family I) or by a big loop (Family II), both stabilized by H‐bonds. Proximity of D‐Ala2‐Phe3‐Asp4and Val5‐Val6‐Gly7triads is an obvious structural similarity between almost all groups in both families of structures. Whereas differences between the two families originated mostly from a transition at Ψ Asp4backbone dihedral angle, the backbone structures at segment 1–4 are similar and spatial arrangements of Tyr1(t) and Phe3(g–) are identical in one group of each family. Moreover, these two groups have a N‐terminal tetrapeptide whose conformation most closely resembles that of a well‐defined group of structures for dermenkephalin. Altogether, these results suggest that conformational attributes that are common to dermenkephalin and deltorphin‐I,i.e.,the backbone conformation of the N‐terminal tetrapeptide and preferential orientations in the side‐chain of Tyr1(t) and Phe3 (g–) underlie their ability to bind with high selectivity to the δ‐opioid receptor.