bluuebunny/crossref_metadata_2025 huggingface dataset:
Pegylated peptides IV
Authors: FELIX ARTHUR M. , LU YI‐AN , CAMPBELL ROBERT M.
Published: August 2010

Conditions have been developed for the site‐specific pegylation (NH2‐terminus, side‐chain and carboxyterminus) of a potent analog of growth hormone‐releasing factor, [Ala15]‐hGRF(1‐29)‐NH2. These pegylated peptides were prepared by solid‐phase peptide synthesis using the Fmoc/tBu strategy, and were fully characterized by analytical HPI.C, amino‐acid analysis,1H‐NMR spectroscopy and laser desorption mass spectrometry. Biological activities of hGRF analogs were determinedin vitroutilizing stimulation of growth hormone release by cultured rat pituitary cells as an index. GH‐releasing potencies of the pegylated hGRF analogs were compared to a series of model analogs of [Ala15]‐hGRF(1‐29)‐NH2that were acetylated or protected as the ethylamides at the pcgylation sites. It was found that acetylation at the NH2‐terminus resulted in reduced potency, which was not further affected when the NH2‐terminus was pegylated, regardless of the size of poly(ethyleneglycol) (PEG) employed (e.g. PEG2000or PEG5000). Pegylation at Asp8or Lys12decreased biological potency, a situation which was exacerbated by increasing the molecular weight of PEG. Pegylation at Lys21or Asp25did not significantly affect biological activity. The C‐terminal model peptide, [Ala15,Orn(Ac)30]‐hGRF(1‐29)‐NH2, was the most potent analog identified in this series (ca. 4–5‐fold that of hGRF(1‐44)‐NH2. The COOH‐terminal pegylated analogs retained this increased level of biological activity independent of PEG molecular weight. These studies demonstrate that a biologically active peptide can be pegylated and retain the fullin vitropotency of the peptide. However, the biological activity is highly dependent on the site of pegylation and, in some cases, the molecular weight of PEG (degree of pegylation) moiety used.
Brain cathepsin B but not metalloendopeptidases degrade rAPP751 with production of amyloidogenic fragments
Authors: MARKS N. , BERG M.J. , SAPIRSTEIN V.S. , DURRIE R. , SWISTOK J. , MAKOFSKE R.C. , DANHO W.
Published: August 2010

Lysosomal cathepsin B but not L degraded rAPP751 to yield C‐terminal 19–25 kDa fragments containingβA4, reinforcing the view that acidic proteases participate in endosonial‐lysosomal processing to yield amyloidogenic fragments in situ. This mechanism is consistent with fragmentation of endogenous APPs within clathrin‐coated vesicles (CVs) by vesicular hydrolases, with the appearance of C‐terminal amyloidogenic fragments following incubation at pH 6.5. A neutral endopeptidase resembling NEP 24.11 (PS‐NEP) purified from detergent extracts of human brain degraded rAPP751; however, breakdown was not blocked robustly by metal chelators or phosphoramidon, suggesting the presence of an alternative processing enzyme. Effects of other inhibitors showed that breakdonn was mediated b) serine‐protease‐like component(s). A phosphoramidon‐insensitive metalloendopeptidase (PI‐NEP) partially purified from rat brain P2 using detergents, and resembling NEP 24.15, showed no activity towards I‐rAPP751.

Peptides containing putative β‐ or γ‐secretase sites were synthesized for purposes of examining their metabolism by the brain enzymes. Those containing β‐secretase sites were hydrolysed at one or more sites by the four enzymes, but only PI‐ and PS‐NEP acted at the Met‐Asp site of Ac‐VaI‐Lys‐Met‐Asp‐Ala‐Glu‐Phe‐Arg.NH2. In the case of substrates Containing the γ‐site, these two categories of enzymes were the only ones degrading N‐Ac‐Ilc‐Ala NH2. These data imply that the brain metalloendopeptidases, while inactive towards intact precursors, may be involved in turnover of intermediates containing β‐ or 7‐sites.
Alkaline protease catalysis of a secondary amine to form a peptide bond
Authors: CHEN SHUI‐TEIN , KAO CHAI‐LIN , WANG KUNG‐TSUNG
Published: August 2010

Procedures for peptide bond formation using esters of l‐amino acids as the acyl donor and secondary amines such as derivatives of d‐ or l‐proline, or pyroglutamic acid as the nucleophile in anhydrous 2‐methyl‐2‐propanol catalyzed by alcalase or subtilisin Carlsberg have been developed. Kinetic studies have shown that all secondary amines tested had catalytic efficiencies (κcat/κm) in the range 84–423 min −1M−1. Both enzymes have identical catalytic properties. The selectivity of the enzyme‐catalyzed reaction in 2‐methyl‐2‐propanol shows that at the s‐1 subsite of the enzyme only l‐amino acids are substrates as acyl donors, and at the s‐1′ subsite both d‐ and l‐amino acids are substrates. Optimal conditions for preparing precursors of the renin inhibitors Z‐Ala‐Pro‐OBzl, Z‐Lys(Z)‐Pro‐OBzl and Z‐Lys(TFA)‐Pro‐OBzl were studied.
Synthesis of α‐factor analogues containing photoactivatable and labeling groups
Authors: JIANG Y. , BRESLAV MICHAEL , KHARE RAM KUMAR , MCKINNEY ANGELA , BECKER JEFFREY M. , NAIDER FRED
Published: August 2010

Analogues of α‐factor, theSaccharomyces cerevisiuetridecapeptide mating pheromone (H‐Trp‐His‐Trp‐Leu‐Gin‐Leu‐Lys‐Pro‐Gly‐Gln‐Pro‐Met‐Tyr‐OH), containing bothp‐benzoyl phenylalanine (Bpa), a photoactivatable group, and 3‐(mono‐ or di‐iodo‐4‐hydroxyphenyl)propanoic acid (iodinated HPP) or biotin as a tag, were synthesized using solid‐phase methodologies on a [phenylacetamidol‐methyl (PAM) resin. Bpa was introduced into the peptides using Bpa‐hydroxybenzotriazole active ester during peptide chain assembly. Biotinylated α‐factor analogues were prepared by assembling the desired peptide on the resin, and then reacting a specific amino group either with the symmetrical anhydride of biotin or with biotin using BOP as the activating agent prior to anhydrous hydrogen fluoride cleavage. Iodinated HPP was incorporated by acylating free peptides with Bolton‐Hunter reagent (3‐[diiodo‐4‐hydroxyphenyl]propanoic acid hydroxysuccinimide ester) inN,N‐dimethylformamide and borate buffer (pH 8.0) solutions. Purification of all peptides to 98% or greater homogeneity was accomplished by high‐performance liquid chromatography on a reversed‐phase μ‐Bondapak C18column with acetonitrile/water/trifluoroacetic acid as the mobile phase. All products were characterized by amino acid analysis and fast atom bombardment mass spectrometry. Two analogues, α‐(diiodotyrosine)‐His‐Bpa‐Leu‐Gln‐Leu‐Arg‐Pro‐Gly‐Gln‐Pro‐Nle‐Tyr‐OH, and ε‐(diiodo‐HPP)‐Lys‐His‐Bpa‐Leu‐Gln‐Leu‐Arg‐Pro‐Gly‐Gln‐Pro‐Nle‐Tyr‐OH, were one‐twentieth to one‐fortieth as active as α‐factor, and exhibited approximately one order of magnitude lower affinity to the α‐factor receptor. The results suggest that these two analogues are α‐factor agonists and that they can be used as probes of the α‐factor receptor. © Munksgaard 1995.
Peptide aggregates: a novel model system to study self‐assembly of peptides
Authors: JAYAKUMAR R. , JAYANTHY C. , GOMATHY L.
Published: August 2010

Ordered aggregates of Val‐Leu‐Pro‐Phe, tetrapeptide 1, have been found in aqueous solutions. Evidence for the formation of aggregates for the above peptide was obtained by conductometric, pH metric, UV and fluorescence spectroscopic techniques. Values of critical micelle concentration (CMC) for the above peptide obtained by these methods are in good agreement with each other. The formation of organized aggregates of the peptide is favoured upon increasing the temperature (viz. the process of aggregation is endothermic). The aggregation number has been determined at different temperatures. Values of ΔG°mΔH°mΔS°m and ΔC°p have also been estimated. Binding studies with the 8‐anilinonaphthyl sulfonic acid (ANS) and pyrene indicate that the interior of the aggregate is nonpolar. There are two processes with regard to the change of thermodynamical parameters like ΔG°mΔH°mΔS°mΔC°p aggregation number (N). In the first process (from 5°C to 40°C) the driving force for aggregation seems to be the positive entropy because of water release due to intermolecular association of ionic moieties. The second process (from 40°C and above) is due to intramolecular ionic interaction. The chemical shifts of the amide protons of the peptide have been presented in the light of inter‐ and intramolecular hydrogen‐bond formation, and forces implicated in aggregation for both the first and second processes. © Munksgaard 1995.
Conformational study of a synthetic analogue of alamethicin Influence of the conformation on ion‐channel lifetimes
Authors: BRACHAIS LAURENT , DAVOUST DANIEL , MOLLE GÉRARD
Published: August 2010

Alamethicin, a 20‐residue peptaibol, induces voltage‐dependent ion channels in lipid bilayers according to the barrel‐stave model. A synthetic analogue (L2) in which all Aib were replaced by Leu shows a conductance behaviour similar to alamethicin, but channel lifetimes are drastically reduced. Among several hypotheses, a different conformation for L2 might be responsible for this phenomenon by increasing the α‐helical content (alamethicin presents some 3.010‐helical parts) and thus decreasing the length of the transmembrane part. A conformational study of L2 was undertaken using FTIR, CD and NMR spectroscopy, and the secondary structure was compared with alamethicin. These techniques showed an enhanced predominant helical structure as compared to alamethicin. Moreover, the NOE pattern showed an exclusively α‐helical conformation, resulting in a smaller length of the L2 peptide. This shortening somewhat impedes the complete crossing of the membrane, and could then explain the reduction of its ion‐channel lifetimes. © Munksgaard 1995.
Single diastereomeric desaminotyrosylalanyl tetra‐ and heptapeptides with opioid antagonistic activity
Authors: BALBONI G. , SALVADORI S. , D'ANGELI F. , MARCHETTI P. , LAZARUS L.H. , BRYANT S.D. , BIANCHI C.
Published: August 2010

The N‐terminal dipeptide Tyr‐d‐Ala of a p‐selective agonist, dermorphin tetrapeptide (DT, H‐Tyr‐d‐Ala‐Phe‐Gly‐NH2) and δ‐selective agonist deltorphin C (DEL‐C, H‐Tyr‐d‐Ala‐Phe‐Asp‐Val‐Val‐Gly‐NH2) was changed into an aminodiacyl moiety. The relevant synthetic step is a nucleophilic substitution of bromine from a chiral 2‐bromopropanamide by the amino group of tyrosine, with overall retention of configuration. The resulting pseudo tetra‐ and heptapeptides I‐VI were characterized for μ and δ‐opioid receptor binding properties using [3H]DAGO and [3H]DPDPE, respectively, and in a bioassay using guinea pig ileum (GPI) and mouse vas deferens (MVD). As a result of chemical alteration of N‐terminal dipeptide moiety, all synthesized analogs showed considerable reduction in opioid receptor affinity compared to μ and δ‐prototypes (500‐fold on the μ‐site, analog I, and 125‐fold on the δ‐site, analog IV). Interestingly, analogs I and IV showed moderate antagonist activity, respectively, on GPI and MVD, with pA2 values of 6.05 and 6.82. Analog IV did not exhibit the δ‐antagonist potency and δ‐selectivity of TIPP peptides. © Munksgaard 1995.
Ras‐dependent maturation of Xenopus oocytes is blocked by modified peptides of GTPase activating protein (GAP)
Authors: LOSARDO JOSEPH E. , HEIMER EDGAR , BEKESI EVA , PRINZO KATHLEEN , SCHEFFLER JULIE E. , NERI ANTHONY
Published: August 2010

Guanosine triphosphatase activating protein (GAP) is an important modulator of p21ras (Ras)‐dependent signal transduction in mammalian cells and in insulin‐induced maturation of Xenopus oocytes. A synthetic octapeptide from the catalytic domain of GAP, residues 899‐906 (F899VFLRLIC906), inhibited GAP‐stimulated hydrolysis of GTP to GDP by Ras in an in vitro biochemical assay (IC50= 12 μM). The peptide was assayed for its ability to block insulin‐ (Ras‐dependent) and progesterone‐ (Ras‐independent) induced maturation of stage VI Xenopus laevis oocytes, marked by germinal vesicle breakdown (GVBD). Microinjection of 50 pmol of the peptide inhibited insulin‐ but not progesterone‐induced GVBD by 50%. A 7‐residue peptide lacking F899, GAP(900‐906)‐NH2, failed to inhibit GAP‐stimulated GTPase activity and did not block GVBD. Replacement of the cysteine residue at position 906 with methionine resulted in a peptide with prolonged inhibitory activity in the oocyte. Moreover, sequential replacement of specific L‐amino acid residues with the corresponding d‐mino acids produced a peptide with a two‐fold increased half‐life after injection into oocytes. None of the peptides tested affected progesterone induced GVBD, suggesting that the modifications did not result in loss of specificity. These studies show that (a) peptides that were able to inhibit GAP‐stimulated Ras GTPase activity in vitro were also able to block Ras‐dependent GVBD in oocytes, and (b) specific substitutions in these peptides can result in improved stability in oocytes. © Munksgaard 1995.
Probing the structure of human growth hormone by limited proteolysis
Authors: DE LAURETO PATRIZIA POLVERINO , TOMA SALVATORE , TONON GIANCARLO , FONTANA ANGELO
Published: August 2010

Digestion of human growth hormone (hGH) with the Glu‐specific protease from Staphylococcus aureus V8 was performed at 20‐22°C or 37°C at a 1:20 ratio (by weight) at pH 7.8 with or without 0.2% SDS. There are 14 Glu‐residues evenly distributed along the polypeptide chain of hGH as possible sites of proteolytic cleavage of V8‐protease. The pattern of fragmentation of hGH was analyzed by electrophoresis and reversed‐phase HPLC, and the identity of the proteolytic fragments isolated to homogeneity was established by their partial sequencing and amino acid analysis after acid hydrolysis. Kinetic analysis of the proteolytic digestion process allowed to establish that initial nicking of the protein occurs at Glu33 and subsequently at Glu56 and Glu66. Much slower cleavages occur at G1u30 and Glu186. These cleavage sites are located at chain loops in the hGH molecule, and in particular outside the helical segments of the four‐helix bundle of the crystal structure of hGH. Fragments 1‐33 and 67‐191 comprising entirely the N‐terminal helix and the three C‐terminal helices of hGH, respectively, were isolated to homogeneity in amounts useful for subsequent conformational and functional studies. The results of this study and of previous ones [Li, C.H. (1982) Mol. Cell. Biochem.46, 31‐41] describing limited proteolysis of hGH by various proteases have been interpreted on the basis of the three‐dimensional structure and dynamics of hGH. Overall, it is shown that proteolytic enzymes preferentially cleave hGH at exposed and flexible loops only, thus emphasizing the fact that proteases can be used as reliable probes of protein structure and dynamics. © Munksgaard 1995.
Synthesis and immunological evaluation of the conjugates composed from a muramyl peptide GMDP and tuftsin
Authors: TITOV VLADIMIR M. , MESHCHERYAKOVA ELENA A. , BALASHOVA TAMARA A. , ANDRONOVA TATYANA M. , IVANOV VADIM T.
Published: August 2010

The conjugates of a muramyl dipeptide analog GMDP (N‐acetylglucosaminyl β1→4 N‐acetylmuramyl‐L‐alanyl‐D‐isoglutamine) and tuftsin (Thr‐Lys‐Pro‐Arg) were synthesized from unprotected GMDP by mixed anhydride procedure. The identity of the conjugates was confirmed by high resolution NMR and their immunomodulating properties were determined in various tests. It was found that the conjugate in which the GMDP carboxyl group forms an amide bond with the &‐amino group of tuftsin lysyl residue exceeds GMDP in all the activities determined. Synergism of GMDP and tuftsin was found in phagocytosis stimulation assay.
Solid‐phase synthesis of oxytocin using iodotrichlorosilane as Boc deprotecting agent
Authors: SIVANANDAIAH K.M. , SURESHBABU V.V. , SHANKARAMMA S.C.
Published: August 2010

Deprotection of the tert‐butoxycarbonyl group during solid‐phase synthesis of peptides can be conveniently and efficiently carried out using a neutral reagent, silicon tetrachloride/sodium iodide (iodotrichlorosilane). This simple and rapid method has been advantageously employed during the solid‐phase synthesis of the pituitary hormone, oxytocin.
Evaluation of the in vitro and in vivo activity of the l‐, d,l‐ and d‐Cit6 forms of the LH‐RH antagonist Cetrorelix (SB‐75)
Authors: PINSKI J. , SCHALLY A.V. , YANO T. , GROOT K. , SRKALOVIC G. , SERFOZO P. , REISSMANN T. , BERND M. , DEGER W. , KUTSCHER B. , ENGEL J.
Published: August 2010

The objective of this study was to examine the in vivo and in vitro gonadotropin‐inhibiting potencies, edematogenic activities and the receptor binding affinities of the d‐Cit6, d,l‐Cit6 and l‐Cit6 forms of the LH‐RH antagonist Cetrorelix (SB‐75) [AC‐d‐Nal(2)1,d‐Phe(4Cl)2,d‐Pal(3)3,d‐Cit6,d‐Ala10]LH‐RH. In order to demonstrate the suppressive effects of two different diastereomers of SB‐75 and their racemic mixture on LH and FSH release, [d‐Cit6] SB‐75 was injected subcutaneously in doses of 2.5 and 10 μg/rat, [d,l‐cit6]‐SB‐75 in doses of 5 and 20 μg/rat and [l‐Cit6] SB‐75 in doses of 12.5 and 50 μg/rat to castrated male rats. Two hours after administration, there was no difference in LH levels between rats injected with the l‐form and control animals, indicating a low activity and/or a rapid enzymatic degradation of this peptide. The (1:1) diastereomeric mixture was only about half as potent in suppression of LH release compared to [d‐cit6] SB‐75. Serum FSH levels were suppressed significantly (p<0.01) for more than 48 h after the administration of 10 μg [d‐Cit6] SB‐75 and 20 μg of [d,l‐Cit6] SB‐75, respectively. [d‐cit6] SB‐75 administered at a dose of 2 μg/rat induced 100% inhibition of ovulation, while 4 μg/rat of the d,l‐Cit6 peptide were necessary to produce the same effect. [l‐Cit6] SB‐75 given at a high dose of 40 μg/rat produced only 14% inhibition of ovulation. The d‐Cit6 form of SB‐75 produced skin lesions with a much smaller diameter than the l‐isomer, and was about 34 times less edematogenic. [d‐Cit6] SB‐75 was bound more powerfully to high‐affinity pituitary LH‐RH receptors than either d,l‐Cit6 or l‐Cit6 analogues. In vitro assays based on the superfusion of dispersed rat pituitary cells on a column, followed by radioimmunoassay for LH, also demonstrated a lower inhibitory activity for the l‐Cit6 analogue, but the differences between d‐, d,l‐ and l‐citrulline analogues were smaller than in vivo. Our results indicate that the LH‐RH antagonist [d‐Cit6] SB‐75 is more effective in suppression of gonadotropin release In vivo and in vitro, less edematogenic and possesses higher binding anity to pituitary LH‐RH receptors than the d,l‐ and l‐citrulline decapeptide analogues. © Munksgaard 1995.
Influence of serine in positionion conformation and dynamics of reverse turns
Authors: MATTER H. , GEMMECKER G. , KESSLER H.
Published: August 2010

NMR spectroscopy has been employed for the conformational analysis of the cyclic hexapeptide cycle(‐d‐Pro1‐Ala2‐Ser3(Bzl)‐Trp4‐Orn5(Z)‐Tyr6‐) with and without protecting groups on Ser3and Orn5. This peptide sequence was derived from the active loop sequence of the α‐amylase inhibitor Tendamistat (HOE 467). The aim was to investigate the role of serine in positioniof a standard β‐turn on the conformation and stabilization of this turn. Based on distance and torsion constraints from 2D NMR spectroscopic measurements in DMSO‐d6solution, structure refinement was accomplished by restrained molecular dynamics (MD) simulationsin vacuoand in DMSO.

The analysis of both structures in solution reveals a considerable effect of the unprotected serine sidechain on the adjacent β‐turn conformation. While in the protected peptide with Ser3(Bzl) a βII‐turn is observed between Trp4and Orn5, the deprotected compound reveals a βI‐turn in this region. The βI‐turn is stabilized by a backbone‐sidechain hydrogen bond from Orn5NαH to Ser3Oγ. Comparisons with other NMR‐derived solution structures of cyclic model peptides and in some protein structures from literature reveal a general structural motif in the stabilization of βI‐turns by serine in theiposition through backbone‐sidechain interactions. © Munksgaard 1995.
Cyclic analogues of Thr6‐bradykinin, N8‐Lys‐bradykinin and endo‐Lys8a‐vespulakinin 1
Authors: GOBBO MARINA , BIONDI LAURA , FILIRA FERNANDO , PIEK TOM , ROCCHI RANIERO
Published: August 2010

Syntheses are described of the endo‐Lys8a‐vespulakinin 1 and of cyclo‐Thr6‐ and cyclo‐Nε‐Lys‐bradykinin. The linear peptides covering the entire sequences of endo‐Lys8a‐VSK‐1 and Thr6‐BK, and the decapeptide containing all residues constituting Lys‐BK, with a Arg‐Lys peptide bond involving the ε‐amino function of lysine, were prepared by the solid‐phase procedure based on Fmoc chemistry. Cyclization was carried out by the diphenylphosphorazide method. The amino‐terminal octapeptide sequence of vespulakinin 1, Fmoc‐Thr(tBu)‐Ala‐Thr(tBu)‐Thr(tBu)‐Arg(Pmc)‐Arg(Pmc)‐Arg(Pmc)‐Gly‐OH, and its Nα‐Boc‐[(Gal β)Thr3, (Gal β)Thr4]‐analogue, were used to prepare Nα‐(1–8 VSK 1)‐cyclo‐Nε‐kallidin and Nα‐[(Gal β)Thr3, (Gal β)Thr4, 1–8 VSK 1]‐cyclo‐Nε‐kallidin. Peptides and glycopeptides were characterized by amino‐acid analysis, optical rotation, analytical HPLC and FAB‐MS. Consistent with previous findings, preliminary pharmacological experiments on smooth muscle preparations showed that the cyclic, or partially cyclic, analogues were significatively less potent than the linear ones. © Munksgaard 1995.
N,N‐Dialkoxycarbonylamino acids from the sodium hydride‐mediated reaction of alkyl chloroformates with mixed anhydrides of N‐alkoxycarbonylamino acids
Authors: BENOITON N. LEO , AKYUREKLI DENIZ , CHEN FRANCIS M.F.
Published: August 2010

Reaction of mixed anhydride R1OCO‐NHCHR2‐CO‐O‐COOR3 for R1= benzyl and tert‐butyl and R3= methyl, ethyl, benzyl and allyl with sodium hydride and R3OCOCI followed by acid hydrolysis gives modest yields of R1OCO‐N(R3OCO)CHR2‐COOH. The products are contaminated by parent acid R1OCO‐NHCHR2‐COOH that is not readily removed. About 75% of the acylation originates from intramolecular transfer of the alkyl carbonate moiety; the remainder comes from acylation by the alkyl chloroformate. © Munksgaard 1995.
Effect of trifluoroethanol on the solution structure and flexibility of desmopressin: a two‐dimensional NMR study
Authors: WANG JIANJUN , HODGES ROBERT S. , SYKES BRIAN D.
Published: August 2010

The solution structures of desmopressin, a nine‐residue peptide with specific antidiuretic and antibleeding activities, have been studied in aqueous and TFE‐containing solutions by two‐dimensional NMR and molecular modeling techniques. It is found that TFE induces a conformational change of the peptide. The structure(s) in water are flexible, and may show multiple conformations, with a significant population of a conformation that contains two fused β‐turns. TFE diminishes the peptide conformational flexibility to form more well defined structure(s). The TFE structure(s) were generated by using molecular modeling based on NOE‐derived distance restraints and hydrogen‐bond restraints obtained from amide proton exchange rates and chemical shift temperature coefficients. While the structure in TFE is more rigid, two different orientations were found for the last two residues in the three residue tail. The conformation of the first seven residues of the peptide is well defined and consists of a short distorted antiparallel β‐sheet with residue Tyr2 and Phe3 in one strand and residue Cys6 and Pro7 in the other strand. A type I β‐turn, centered in residues Gln4 and Asn5, connects the two strands. A distorted type II β‐turn is found in the three‐residue tail involving residues Cys6‐Pro7‐d‐Arg8‐Gly9 in both families of TFE structures. © Munksgaard 1995.
In vitro association of the phosphatidylinositol 3‐kinase regulatory subunit (p85) with the human insulin receptor
Authors: OTTINGER ELIZABETH A. , HUI TO YUEN , MAN ZHIJUN , BARANY GEORGE , BERNLOHR DAVID A.
Published: August 2010

The insulin receptor, as a consequence of ligand binding, undergoes autophosphorylation of critical tyrosyl residues within the cytoplasmic portion of its β‐subunit. The 85 kDa regulatory subunit of phosphatidylinositol (PI) 3‐kinase (p85), an SH2 domain protein, has been implicated as a regulatory molecule in the insulin signal transduction pathway. For the present study, glutathione S‐transferase (GST) fusion proteins of p85 SH2 domains were used to determine if such motifs associate directly with the autophosphorylated human insulin receptor. The p85 N + C (amino plus carboxyl) SH2 domains were demonstrated to associate with the autophosphorylated β‐subunit, while neither the GTPase activator protein (GAP) N SH2 domain nor the phospholipase C‐γ1 (PLCγ1) N + C SH2 domains exhibited measurable affinity for the activated receptor. The p85 N SH2 domain demonstrated weak association with the insulin receptor, while the p85 C SH2 domain alone formed no detectable complexes with the insulin receptor. The association of p85 N + C SH2 domains with the autophosphorylated receptor was competed efficiently by a 15‐residue tyrosine‐phos‐phorylated peptide corresponding to the carboxyl‐terminal region of the insulin receptor, but not by phos‐phopeptides of similar length derived from the juxtamembrane or regulatory regions. The insulin receptor C domain phosphopeptide inhibited the p85 N i C SH2 domain‐insulin receptor complex with an IC0.5 of 2.3 ± 0.35 μM, whereas a 10‐residue phosphopeptide derived from the insulin receptor substrate 1 (IRS‐1) competed with an IC0.5 of 0.54 ± 0.10 μM. These results demonstrate that, in vitro, there is an association between the p85 regulatory protein and the carboxyl‐terminal region of the activated insulin receptor that requires the presence of both the N and C SH2 domains. Furthermore, formation of the p85/insulin receptor complex may lead to signaling pathways independent of IRS‐1. © Munksgaard 1995.
Stereochemical analysis of the antigenic tip of the V3 loop peptide of HIV‐1 gp120
Authors: GUNASEKARAN K. , RAMAKRISHNAN C. , BALARAM P.
Published: August 2010

A novel multiple turn conformation has been observed for a segment GPGRAFY in the crystal structure of a complex of HIV‐1 gp120 V3 loop peptide with the Fab fragment of a neutralizing antibody [Ghiara et al. (1994) Science264, 82–85]. A structural motif has been defined for the peptide segment, employing idealized backbone conformations characterized by ranges of virtual Cα torsion angles and bond angles. A search of 122 high‐resolution protein crystal structures has permitted identification of 24 examples of similar structural motifs. Two major conformational families have been identified, which differ primarily in the conformation at residue 3. The observed conformation at residue 3 in family 1 is left‐handed helical (αL) and that in family 2 is right‐handed helical (αR). Of the 10 examples in family 1, 9 examples have Gly residues at position 3. Of the 12 examples in family 2, 7 examples have Asn/Asp at position 3. Computer modeling of the V3 loop tip sequence using the two backbone conformational families as starting points leads to minimum‐energy conformations in which antigenically important side‐chains occupy similar spatial arrangements. This stereo‐chemical analysis of the V3 loop tip sequence suggests a rational basis for the design of synthetic analog peptides for use as viral antagonists or synthetic antigens. © Munksgaard 1995.
Isolation and identification of C‐terminal peptides of proteins
Authors: GU QU‐MING , SIH CHARLES J.
Published: August 2010

A method for the isolation and identification of C‐terminal peptides of proteins has been developed. The procedure entails the racemization of the C‐terminal amino acid by reaction of the N‐trifluoroacetylated protein with acetic anhydride and pyridine. After deprotection, the protein is fragmented to yield a mixture of peptides, which in turn are digested with carboxypeptidases. All peptides are hydrolyzed to L‐amino acids except the C‐terminal peptide with the terminal D‐amino‐acid residue. It is resistant to the action of carboxypeptidases and is readily identified by peptide mapping. © Munksgaard 1995.
Conformation and activity of +H2‐Pro‐Leu‐Gly‐NH2 and its analogues modified at the leucyl residue
Authors: KIM YOUNG JU , HAN SEONG JUN , KIM SHI CHOON , KANG YOUNG KEE
Published: August 2010

In order to investigate the relation between the conformation and the ability to modulate dopamine receptors, conformational free‐energy calculations using an empirical potential (ECEPP/3) and hydration shell model were carried out on the tripeptide +H2‐Pro‐Leu‐Gly‐NH2 (PLG) and its analogues in which the Leu residue is replaced with Phe, Ahx (L‐2‐aminohexanoic acid), Ile, Abu and Ala residues in the unhydrated and hydrated states. PLG and two tripeptides possessing Phe and Ahx residues show dopamine receptor modulating activity, while the other tripeptides do not. Irrespective of activities, PLG and its analogues are found to have the high probabilities to form β‐bends of types II and I in the unhydrated state. However, in the hydrated state, the β‐bend probabilities of the PLG analogues decrease significantly compared with those in the unhydrated state. These results indicate that the β‐bend structure is a necessary factor for the PLG analogues to be active, but not a sufficient factor, and that the interactions of water molecules with the backbone may force the tripeptides to be more distorted or extended. The size and the orientation of the hydrophobic moiety of the second residue seem to be of consequence in eliciting the activity of the tripeptides. © Munksgaard 1995.
Solution structure of the B‐chain of insulin as determined by 1H NMR spectroscopy Comparison with the crystal structure of the insulin hexamer and with the solution structure of the insulin monomer
Authors: HAWKINS BERNADEITE , CROSS KEITH , CRAIK DAVID
Published: August 2010

The solution structure of the isolated B‐chain of bovine insulin has been determined by 1H NMR spectroscopy combined with simulated annealing calculations. Complete sequence‐specific assignments for the proton resonances are reported together with a set of 309 NOES used in the structure calculations. Chemical‐shift variations from random coil values provide support for the existence of helical regions in the polypeptide chain, as do a characteristic series of dαp(i,i+ 3) NOES from residues B8 to B17. While there is some evidence for a limited degree of conformational averaging over the helical region, in general the helix is relatively well defined and corresponds closely to the helical region seen in the X‐ray crystal structure of the insulin hexamer. Other similarities with the crystal structure include turn‐like conformations at the carboxy terminal end of the helix and extended strands at both the amino and carboxy termini of the peptide. These similarities between the crystal structure and the isolated B‐chain suggest that this peptide has intrinsic folding properties, which allow it to adopt its characteristic structure in intact insulin without the need for extensive cooperative interactions with the A‐chain. Despite these general similarities, an important difference between the isolated B‐chain and the intact protein occurs in the carboxy terminal region. This region appears significantly more mobile in the isolated B‐chain. As a conformational change involving the carboxy terminus has been implicated in receptor binding, the current study of the isolated B‐chain provides valuable information on the extent of this region's intrinsic mobility. © Munksgaard 1995.
Linear and cyclic synthetic peptides related to the main autophosphorylation site of the Src tyrosine kinases as substrates and inhibitors of Lyn †
Authors: RUZZA PAOLO , CALDERAN ANDREA , FILIPPI BRUNO , BIONDI BARBARA , DEANA ARIANNA DONELLA , CESARO LUCA , PINNA LORENZO A. , BORIN GIANFRANCO
Published: August 2010

Tyrosine protein kinases (TPKs) of the src family contain two major phosphoacceptor sites which are homologous to the Tyr 416 and Tyr 527 of pp60c‐src. The former represents the main autophosphorylation sites of these enzymes, and its phosphorylation correlates with increased kinase activity.

It has previously been demonstrated that the Src‐like tyrosine kinase expressed by the oncogene lyn displays a high affinity toward the heptapeptide H‐Glu‐Asp‐Asn‐Glu‐Tyr‐Thr‐Ala‐OH, which reproduces the main autophosphorylation site of the Src family enzymes [Donella‐Deana, A., Marin, O., Brunati, A.M. & Pinna, L.A. (1992) Eur. J. Biochem.204, 1159–1163].

Our study was addressed to the synthesis of some derivatives of this sequence in order to obtain both peptide substrates suitable for the detection of the Src‐like tyrosine kinase activity and active site‐directed inhibitors specific for this class of enzymes. For this purpose we synthesized by classical solution methods the heptapeptide and its dimeric form. Moreover, in order to improve the proteolytic resistance of these peptides we also synthesized their cyclic derivatives and their N‐terminal acetylated and C‐terminal amidated analogs. The correlation between the different structural properties induced by the modifications of the native sequence and the propensity of the peptides to act as Lyn substrates was examined. The kinetic data obtained indicate that the extent of the peptide phosphorylation varies considerably depending on the flexibility and length of the analogs. While the cyclization and the C‐terminal amidation of the heptapeptide are detrimental for the Lyn activity, dimeric derivatives display very favourable kinetic constants. In particular the cyclic dimer is an especially suitable substrate for the tyrosine kinase and a powerful inhibitor of both the phosphorylation activity of Lyn and the enzyme autophosphorylation. © Munksgaard 1995.
Drastic reduction of antimicrobial activity by replacement of Orn residues with Lys in cyclized amphiphilic β‐structural model peptides
Authors: ANDO SETSUKO , NISHIHAMA MASANORI , NISHIKAWA HIROSHI , TAKIGUCHI HIDEO , LEE SANNAMU , SUGIHARA GOHSUKE
Published: August 2010

Recent investigations have indicated that cyclic dodeca‐ and tetradecapeptides, Cyclo(‐LeU‐Orn‐Leu‐Orn‐D‐Phe‐Pro‐)2 (Orn‐DLL‐12) and cyclo(‐Leu‐Orn‐Leu‐Orn‐Leu‐D‐Phe‐Pro‐)2 (Orn‐DLL‐14), which are designed on the basis of a cyclic β‐structural antibiotic, gramicidin S (GS), inhibit the growth of Gram‐positive and ‐negative bacteria with high potency [Ando, S., Nishikawa, H., Takiguchi, H., Lee, S. & Sugihara, G. (1993) Biochim. Biophys. Acta 1147, 42‐49]. In this study we designed and synthesized two analogs, Lys‐DLL‐12 and Lys‐DLL‐14, in which four Orn residues in Orn‐DLL‐12 and Om‐DLL‐14 were replaced by Lys residues, respectively, and investigated their interactions with model membranes in terms of CD and dye‐leakage experiments, antimicrobial activity and lytic activity for human erythrocytes. Both peptides newly designed showed no antimicrobial activity and no lytic activity for erythrocytes. The present CD study showed that the presence of neutral liposomes and of acidic liposomes of natural or synthetic phospholipids results in no remarkable conformational difference between Orn‐DLL‐12/‐14 and Lys‐DLL‐12/‐14. The leakage experiment showed a clear relation between the antimicrobial activity and the leakage ability in acidic synthetic phospholipid liposomes but no correlation in acidic natural ones. The difference in hydrophobic and hydro‐philic balance between Orn‐DLL‐12/14 and Lys‐DLL‐12/14 (derived from the increasing hydrophobicity due to an increase of four methylene units by the substitution of Lys for Orn) may be one of the important factors in the drastic decrease in activity. © Munksgaard 1995.
Effector‐induced dissociation of glyceraldehyde‐3‐phosphate dehydrogenase discriminated by urea solvation
Authors: IVANOVA VESSELINA , KRUSTEVA NIKOLINA , ATANASOV BORIS
Published: August 2010

The dissociation of glyceraldehyde‐3‐phosphate dehydrogenase (GAPD) from pig muscle in water solutions (0.1 M phosphate, pH 7) at increased urea concentrations was studied by means of frontal‐gel chromatography, intrinsic (TRP) fluorescence, differential absorption spectroscopy and selective chemical modification at TRP‐193. The results are in agreement with a consecutive two‐step model of dissociation of the tetramer and the dimer (C*T= 0.42 M urea < C*D= 1.39 M urea). The binding of effector(s) destabilizes the oligomeric structures (ΔGT changes from ‐1.00 to ‐0.54 kcal/mol; ΔGD from ‐2.30 to ‐1.22 kcal/mol). The introduction of the bulky Koshland‐reagent group to TRP‐193 at the subunit‐subunit interface leads to a decrease of the stability with δΔG ≅ 1 kcal/mol, owing to TRP‐193…TYR‐39…TYR‐92 cluster destruction. By using lobster GAPD atomic coordinates (PDB file 1GPD) and pig muscle GAPD amino‐acid sequence, a tentative molecular model was constructed and the subunit contacts in terms of the Lee‐Richard static accessibilities were described. A detailed analysis of the dissociation as a transfer of the buried residues from the molecular interface to the urea solutions was performed.
New cyclic bradykinin antagonists containing disulfide and lactam bridges at theN‐terminal sequence*
Authors: SEYFARTH L. , PINEDA DE CASTRO L. FELIPE , LIEPINA I. , PAEGELOW I. , LIEBMANN C. , REISSMANN S.
Published: August 2010

Continuing our studies of the bioactive conformation of bradykinin (BK) antagonists, we synthesized a first series of analogues with side‐chain cyclization in theN‐terminal sequence. Through this conformational constraint it should be possible to gain insight into their three‐dimensional structure. The cycles were proposed on the basis of existing ideas and hypotheses about the receptor bound conformation of BK and its antagonists. The reported peptides contain D‐Phe at position 7 or D‐Tic‐Oic (D‐tetrahydroisoquinoline‐3‐carboxyl‐octahydroindole‐2‐carboxylic acid) at positions 7 and 8, respectively, and a disulfide or lactam bridge between positions 0 and 6. Syntheses, including cyclization reactions, were carried out on PAM resin. The biological activity of the lead compound [Dphe7]‐BK, the linear precursors and the cyclic peptides, as estimated on isolated rat uterus, guinea pig ileum and lung strips, are in the same range. The conformational properties of the new cyclic analogues were studied through energy minimization on a model compound. The results of the calculations support the existence of low‐energy structures containing a β‐turn. Therefore, such a turn in the N‐terminal segment of the molecule can be proposed as an important structural feature of the bioactive conformation of BK antagonists. © Munksgaard 1995.
Synthesis of peptide amides using Fmoc‐based solid‐phase procedures on 4‐methylbenzhydrylamine resins
Authors: THOMPSON PHILIP E. , KEAH HOOI H. , GOMME PETER T. , STANTON PETER G. , HEARN MILTON T.W.
Published: August 2010

A two‐step low‐high protocol for the efficient synthesis of peptide amides is described. The protocol exploits the efficiency of Reagent K for side‐chain deprotection with the capability of the hard acid trifluoromethane‐sulfonic acid (TFSMA) for cleavage of the peptide from the benzhydrylamine resin. This procedure has proven to be an effective method for the synthesis of peptide amides. The formation of α‐aminosuccinimide (Asu) derivatives were observed with aspartyl‐containing peptides as a minor side reaction product of this procedure, but this Asp→Asu rearrangement could be successfully suppressed by employing low temperature conditions. The N‐ to O‐acyl rearrangement of threonine and/or serine residues also only occurred to a minor extent under these synthetic conditions. © Munksgaard 1995.
Engineering of five 88‐residue receptor‐adhesive modular proteins containing a parallel α‐helical coiled coil and two RGD ligand sites
Authors: SLATE CHERYL A. , WENINGER STACIE C. , CHURCH FRANK C. , ERICKSON BRUCE W.
Published: August 2010

Several 88‐residue proteins were designed, synthesized and examined as receptor‐adhesive modular proteins (RAMPs). Three covalent and two noncovalent dimers were made from two 44‐residue peptide chains containing three structural modules: RGD‐A23a (ligand‐spacer‐coil) and A9a‐RGD (coil‐spacer‐ligand). The ligand module contained the tripeptide Arg‐Gly‐Asp (RGD). The coil modules A9a and A23a were five‐heptad α‐helices engineered by Hodges and co‐workers [Int. J. Peptide Protein Res. (1992) 40, 171–1791, By circular dichroic spectroscopy, each of these five RAMPs contained an α‐helical coiled coil. The disulfide‐bridged dimer RGD‐A23a/RGD‐A23a and its reduced form (RGD‐A23a)2 had two N‐terminal RGD sites. The disulfide‐bridged dimer A9a‐RGD/A9a‐RGD and its reduced form (A9a‐RGD)2 had two C‐terminal RGD sites. However, the disulfide‐bridged heterodimer RGD‐A23a/A9a‐RGD had one RGD site at each terminus with a 50 Å coiled coil between them. The temperature at the midpoint of unfolding for each of the covalent homodimers RGD‐A23a/RGD‐A23a (67°C) and A9a‐RGD/A9a‐RGD (69°C) was slightly higher than that of the corresponding noncovalent homodimer (RGD‐A23a)2 (62°C) or (A9a‐RGD)2 (68°C) but much lower than that of the covalent heterodimer RGD‐A23a/A9a‐RGD (79° C). The enthalpy and entropy of thermal unfolding were also significantly greater for the heterodimer than for the four homodimers, consistent with the heterodimer having the most stable coiled coil. Although the distance between its RGD sites was at least 50 A greater than that for the homodimers, this heterodimeric RAMP was only as active as the homodimers A9a‐RGD/A9a‐RGD and (A9a‐RGD)2 in inhibiting the adhesion of A2058 melanoma cells to extracellular matrix proteins. Thus the coiled‐coil heterodimeric RAMP RGD‐A23a/A9a‐RGD was unable to bind to two integrin receptors on the same cell at the same time. The Hodges coiled coil A23a/A9a was useful as an oriented pair of coil modules for placing ligand modules at the opposite ends of an elongated RAMP.
Chemical shift differences between free and Fab‐bound peptide correlate with a two‐stage selection of peptide sequences from a random phage display library to delineate critical and non‐critical residues for antibody recognition
Authors: STOCKMAN BRIAN J. , BANNOW CAROL A. , MICELI ROBERT M. , DEGRAAF MICHAEL E. , FISCHER H. DAVID , SMITH CLARK W.
Published: August 2010

Epitope libraries provide a method to identify peptide ligands for antibodies, receptors or other binding proteins. As such, they provide a powerful tool to rapidly identify lead ligands in the drug discovery process. In an attempt to correlate structural information with the results from peptide screening, we have used NMR spectroscopy of peptide/antibody complexes to demonstrate that core residues identified through a two‐stage selection process undergo a larger structural change upon binding antibody than do positions in the peptide amenable to a variety of side chains. The model system used was the M2 monoclonal antibody/Flag™ octapeptide epitope system. We have analyzed two peptides: Ac‐Asp‐Tyr‐Lys‐Leu‐Gly‐Asp‐Asp‐Leu‐NH2 (peptide l), which contains several non‐core positions randomized, and Ac‐Asp‐Tyr‐Lys‐Asp‐Asp‐Asp‐Asp‐Leu‐NH2 (peptide 2), which closely corresponds to the original Flag™ sequence. Enrichment of the peptides with 15N facilitated the investigation by permitting spectral editing of the peptide resonances in the presence of antibody. For peptide 1 the absolute shifts for the free vs. Fab‐bound peptide were found to be largest for the amide groups of Asp‐1 and Asp‐6, in agreement with classification of these residues as critical by the phage display library selection process. For peptide 2 the largest absolute shifts were observed for Asp‐1 and Asp‐4, with the other aspartic acid residues also showing significant but smaller changes. © Munksgaard 1995.
Structural characterizations of neuropeptide tyrosine (NPY) and its agonist analog [Ahx5‐17]NPY by NMR and molecular modeling
Authors: BOULANGER YVAN , CHEN YANMIN , COMMODARI FERNANDO , SENÉCAL LOUIS , LABERGE ANNE‐MARIE , FOURNIER ALAIN , ST‐PIERRE SERGE
Published: August 2010

The structures of human NPY and of its centrally truncated agonist analog [Ahx5–17]NPY have been investigated in DMSO‐d6by two‐dimensional NMR and by molecular modeling. For both peptides, a complete resonance assignment was achieved and a large number (more than 200) of inter‐residue NOE connectivities were observed, including long‐range connectivities between theN‐ andC‐terminal ends of the chain. Molecular models were calculated using NOE constraints by distance geometry, simulated annealing and conjugate gradient energy minimization. The results indicate that both peptides are folded in the center of their chain, NPY adopting the hairpin shape, whereas the central portion of [Ahx5–17]NPY is characterized by relatively large loops. In contrast to previous models, practically no α‐helical structure exists for these peptides under our conditions, but two β‐turns are found in NPY and one in [Ahx5–17]NPY. The proximity of the terminal ends could be the determinant factor for their activity. © Munksgaard 1995.
Structural effects of the selective reduction of amide carbonyl groups in motilin 1–12 as determined by nuclear magnetic resonance
Authors: BOULANGER YVAN , KHIAT ABDESSLEM , CHEN YANMIN , GAGNON DIANE , POITRAS PIERRE , ST‐PIERRE SERGE
Published: August 2010

Motilin is a 22‐residue peptide stimulating stomach and intestinal motility. The motilin 1–12 fragment displays biological effects similar to the native peptide. Selective reduction of the amide carbonyl groups to form CH2NH analogs leads to a significant reduction in activity for the first two N‐terminal positions and to a complete loss of activity for all other positions. The structures of motilin 1–12 and ten reduced analogs were investigated using the temperature dependence of the amide NH chemical shifts. In all the analogs, the structure of the N‐terminal region (residues 1–5) was different from the structure of motilin 1–12, which is characterized by hydrogen bonding between Phe1 and Ile4. The structure of the C‐terminal region of analogs was similar to the structure of moth 1–12 for the first two reduction positions only (1–2 and 2–3), indicating that the C‐terminal portion of motilin 1–12 is more critical for biological activity. Complete structural characterizations of motilin 1–12, [CH2NH]1–2, and [CH2NH]4–5‐motilin 1–12 were performed by two‐dimensional NMR spectroscopy and molecular modeling. The structural features observed confirm the differences based on the temperature dependence of the amide NH chemical shifts. These results demonstrate that conservation of the amide bond rigidity is essential for the activity of non‐hydrolyzable analogs. © Munksgaard 1995.
Chemically derived peptide libraries: A new resin and methodology for lead identification
Authors: BUETTNER JOSEPH A. , DADD CHRISTOPHER A. , BAUMBACH GEORGE A. , MASECAR BARBARA L. , HAMMOND DAVID J.H.
Published: August 2010

We have developed a new resin for peptide synthesis that can be used to synthesize and evaluate directly combinatorial peptide libraries for binding target proteins. Fidelity of the peptide synthesis using this hydrophilic resin is comparable to polystyrene‐based resins. Peptide libraries synthesized on this resin were probed by a two color PEptide Library Immunostaining Chromatographic ANalysis (PELICAN) technique for sequences binding the serine protease Factor IX zynogen. This PELICAN technique readily distinguishes between beads interacting with the reagents for target detection (blue beads) from those beads specific for the target protein itself (red beads). Validation of the PELICAN technique, as well as purification of Factor IX from plasma, is demonstrated utilizing this resin. ©Munksgaard 1996.