bluuebunny/crossref_metadata_2025 huggingface dataset:
Transglutaminase‐catalyzed modifications of SV‐IV, a major protein secreted from the rat seminal vesicle epithelium
Authors: PORTA R. , ESPOSITO C. , GENTILE V. , MARINIELLO L. , PELUSO G. , METAFORA S.
Published: August 2010

One of the major proteins secreted from the rat seminal vesicle epithelium, namely SV‐IV, was shown to act in vitro as acyl donor and acceptor substrate for transglutaminase from both guinea pig liver and rat anterior prostate secretory fluid. Electrophoretic and chromatographic experiments indicated that the enzyme catalyzed the formation of multiple modified forms of SV‐IV. In the absence of small Mr amines, transglutaminase was able to produce at least six different molecular forms of the protein, half of which possessed an Mr higher than that of native SV‐IV. These findings suggested that a variable number of intermolecular, and perhaps intramolecular, crosslinks were formed between one or both glutamine residues and one or more lysine residues occurring in the SV‐IV polypeptide chain. In addition, at least three modified forms of the protein were produced by transglutaminase in the presence of high concentrations of spermidine, thus indicating the formation of different (γ‐glutamyl) polyamine derivatives of SV‐IV. Rabbit uteroglobin and rat anterior prostate secretory protein(s) were also shown to be able to covalently bind spermidine in the presence of the enzyme. The possible biological significance of transglutaminase‐mediated modifications of SV‐IV, as well as of other proteins occurring in the mammal seminal fluid, are discussed.
Parallel and antiparallel aggregation of α‐helices
Authors: KARLE ISABELLA L. , FLIPPEN‐ANDERSON JUDITH L. , SUKUMAR M. , BALARAM P.
Published: August 2010

An apolar helical decapeptide with different end groups, Boc‐ or Ac‐, crystallizes in a completely parallel fashion for the Boc‐analog and in an antiparallel fashion for the Ac‐analog. In both crystals, the packing motif consists of rows of parallel molecules. In the Boc‐crystals, adjacent rows assemble with the helix axes pointed in the same direction. In the Ac‐crystals, adjacent rows assemble with the helix axes pointed in opposite directions. The conformations of the molecules in both crystals are quite similar, predominantly α‐helical, except for the tryptophanyl side chain where χ1≃ 60° in the Boc‐ analog and ≃ 180° in the Ac‐analog. As a result, there is one lateral hydrogen bond between helices, N(lε)…O(7), in the Ac‐analog. The structures do not provide a ready rationalization of packing preference in terms of side‐chain interactions and do not support a major role for helix dipole interactions in determining helix orientation in crystals. The crystal parameters are as follow. Boc‐analog: C3H7,N11O13 C3H7OH, space group P1 with a = 10.250(3) Å, b = 12.451(4) Å, c = 15.077(6) Å, x= 96.55(3)°, β= 92.31(3)°, y= 106.37(3)°, Z = 1, R = 5.5% for 5581 data (|F| > 3.0a(F)), resolution 0.89 Å. Ac‐analog: C57,H91,N11,O12, space group P21, with a = 9.965(1) Å, b = 19.707(3) Å, c = 16.648(3) Å, β= 94.08(1), Z = 2, R = 7.2% for 2530 data (|F| > 3.0σ(F)), resolution 1.00 Å.
Structural transition of bovine plasma albumin in the alkaline region the N‐B transition
Authors: ERA SEIICHI , ITOH KIYOOMI B. , SOGAMI MASARU , KUWATA KAZUO , IWAMA TORU , YAMADA HIROMU , ATARI HIROSHI W
Published: August 2010

Bovine plasma albumin (BPA) has approximately one SH group (Cys‐34) which catalyzes the intramolecular SH, S‐S exchange reaction in the alkaline region at low ionic strength, resulting in the formation of the aged form. So, the N‐B transition at ionic strength above 0.20 and below 0.10 was studied using BPA and iodoacetamide‐blocked BPA (IA‐BPA), respectively. (1) pH profiles of [θ]262 and [θ]268 of BPA in 0.20m KCl showed the characteristic changes in the pH region 7.0‐9.0, corresponding to the N‐B transition. On going from pH 7.0 to 9.0 in 0.10 M KCl or NaCl, IA‐BPA did not show significant changes in rotational relaxation times of tryptophyl fluorophors, CD‐resolved secondary structures, spin‐echo 1H‐n.m.r. spectra and cross‐relaxation times (T1S) between irradiated and observed protein protons, which might reflect the rigidity of the domains and/or subdomains. On the other hand, rotational relaxation times of l‐anilino‐8‐naphthalenesulfonate‐IA‐BPA complex (IA‐BPA‐ANS0 9, molar ratio of ANS to IA‐BPA = 0.9/1) showed significant decreases from 131 to 114ns on going from the N‐ to the B‐forms in 0.10M KCl. The above results and reported experimental evidence might indicate that on going from the N‐ to the B‐forms in 0.10 m KC1 or NaCl, the mutual movement of subdomains, connected with a flexible hinge region (Brown & Shockley (1982)) might increase without loss in the helicity and the rigidity of subdomains. (2) The N‐B transition of IA‐BPA in the absence of salt was quite different from those in 0.10 M KCl or NaCl. Decreases in the helicity and the intramolecular rigidity, as monitored by TIS‐measurements, were observed on going from the N‐ to the B‐forms.
Design, synthesis, and biological activities of a potent and selective α‐melanotropin antagonist
Authors: AL‐OBEIDI FAHAD , HRUBY VICTOR J. , HADLEY MAC E. , SAWYER TOMI K. , DEL CASTRUCCI ANA M.
Published: August 2010

Based on structure‐activity relationships of the potent α‐MSH agonist, Ac‐Nle4‐Asp5‐His6‐d‐Phe7‐Arg8‐Trp9‐Lys10‐NH2, several analogs of the general formula Ac‐Nle4‐Asp5‐Waa6‐Xaa7‐Yaa8‐Zaa9‐Lys10‐NH2 were synthesized and tested on frog and lizard skin bioassays for their possible inhibitory actions against α‐MSH on melanocyte stimulation. When Waa6= Trp, Xaa7= D‐Phe, Yaa8= Nle and Zaa = Trp, a highly potent α‐MSH antagonist, Ac‐Nle‐Asp‐Trp‐d‐Phe‐Nle‐Trp‐Lys‐NH., with selectivity on the frog skin α‐MSH receptor system (PA2= 8.4) was obtained. However, several modifications in the amino acid sequence of the peptide resulted in a complete loss of antagonistic activity and a recovery of very weak agonistic action. The following changes in the amino acid sequence of the peptide were examined; His or d‐Trp for Waa, l‐Phe for Xaa, Arg, Ala or Pro for Yaa, and d‐TV for Zaa. All resulted in full agonists with no antagonistic activity. In addition, lactam cyclization between the Asp5 and Lys10 side chains in the antagonist gave a full agonist and a complete loss of antagonistic activity. Efforts to develop a rational approach for the design of selective α‐MSH antagonists for the frog skin α‐MSH receptor will be discussed.
Evidence of neuropeptide bivalency in the interaction of substance P with cerebroside sulfate
Authors: RAO A.G. APPU , CANN JOHN R. , STEWART JOHN M. , WINZOR DONALD J.
Published: August 2010

A precipitin effect has been observed with mixtures of cerebroside sulfate and the neuropeptide substance P. This phenomenon is attributed to multivalency of the lipid due to its existence in micellar form, and to bivalency of substance P. One of those neuropeptide sites is almost certainly the basic residue(s) located at the N‐terminal of substance P, whereas the hydrophobic residues at the C‐terminus are suggested as candidates for the other site on the basis of turbidimetric, circular dichroic, and fluorometric studies. An intrinsic asSociation constant of 3.6 × 104 M‐1 has been obtained from the cerebroside sulfate concentration asSociated with maximal turbidity of mixtures containing a fixed concentration of the neuropeptide.
Facile SPS of peptides having C‐terminal Asn and Gln*
Authors: BREIPOHL GERHARD , KNOLLE JOCHEN , STÜBER WERNER
Published: August 2010

Attachment of Fmoc‐asparagine or glutamine to p‐alkoxybenzyl alcohol type resins has always been difficult and not very effective. A very simple and effective method for the preparation of peptides terminating in asparagine or glutamine is described. The method involves quantitative attachment of Fmoc‐Asp‐OtBu or Fmoc‐Glu‐OtBu via their side‐chain carboxyl group to a resin functionalized with our TMBPA linker for peptide amides. Peptide synthesis is performed using our standard Fmoc chemistry, and treatment with acid, e.g. TFA/DCM/scavenger mixtures, releases the Asn or Gln peptides.
Conformation of a cyclic decapeptide analog of a repeat pentapeptide sequence of elastin: cyclo‐bis(valyl‐prolyl‐alanyl‐valyl‐glycyl)
Authors: BHANDARY KRISHNA K. , SENADHI SHOBHA E. , PRASAD KARI U. , URRY DAN W. , VIJAY‐KUMAR SENADHI
Published: August 2010

The conformation of a cyclic decapeptide analog of a repeat sequence of elastin has been determined in the crystalline state using X‐ray crystallographic techniques. Tetragonal crystals were grown from a solution of the decapeptide in water; space group P42212, a = 19.439(2) & c = 13.602(1) Å, with four formula units (C40H66N10O10·4H2O) per unit cell. The cyclic decapeptide in the crystal exhibits exact twofold symmetry. The asymmetric unit contains one pentapeptide and two water molecules for a total of 32 nonhydrogen atoms. The structure has been determined by the application of direct methods and refined by full‐matrix least squares to an R index of 0.053 for 2272 reflections with intensities greater than 2σ(I). The backbone conformation of the asymmetric pentapeptide can be described as consisting of a double β bend of Type III‐I. The Type III turn has Pro (ρ= ‐59.3°, Ψ= ‐26.8°) and Ala (ρ= ‐65.9°, Ψ= ‐23.1°) at the corners while Type I turn has Ala (ρ= ‐65.9°, Ψ= ‐23.1°) and Val (ρ= ‐98.9°, Ψ= 8.3°) as the corner residues. The cyclic decapeptide has two such double bends linked together by Gly‐Val bridges.
Structure of the fibrinogen binding sequence: arginylglycylaspartic acid (RGD)
Authors: EGGLESTON DRAKE S. , FELDMAN SCOTT H.
Published: August 2010

The crystal structure of a tetrahydrated form of l‐arginyl‐glycyl‐l‐aspartic acid (RGD), the consensus sequence for binding of fibrinogen to cell surface receptors, has been determined from diffractometer data. The tripeptide was crystallized in double zwitterionic form via hanging drop vapor diffusion experiments at a pH near 6.5. The orthorhombic unit cell contains four formula units in space group P2121 21 with lattice parameters a = 4.852(4), b = 11.376(3), c = 34.083(8) Å at RT. The structure was solved by direct methods and refined to a final R = 0.067 based upon 1345 observations with I 2σ(1). Peptide bonds both are trans, ω2= 174.2(6)° and ω3= ‐ 169.3(6)°. The backbone bends at glycine with ρ2= ‐85.5(8)°. One of the water molecules sits between the arginyl side chain and the C‐terminal carboxylate, forming an intramolecular hydrogen bond to the glycyl carboxyl and linking adjacent molecules through two other H‐bond interactions. Comparison of the structure to RGD sequences extracted from 3‐D protein structures reveals a diversity of conformations for this tripeptide sequence.
Conformational features responsible for the binding of cyclic analogues of enkephalin to opioid receptors
Authors: NIKIFOROVICH GREGORY V. , GOLBRAIKH ALEXANDER A. , SHENDEROVICH MARK D. , BALODIS JURIS
Published: August 2010

Models of μ‐ and δ‐receptor‐bound backbone conformations of enkephalin cyclic analogues containing Phe4 were determined by comparing geometrical similarity among the previously found low‐energy, backbone structures of ‐enkephalinamide, ‐enkephalinamide, ‐enkephalin and ‐enkephalin. The present μ‐receptor‐bound conformation resembles a β‐I bend in the peptide backbone centred on the Gly3‐Phe4 region. Two slightly different models were found for the δ‐receptor‐bound conformation; both of them are more extended than the μ‐receptor‐bound conformation and include a γ‐turn (or a γ‐like turn) on the Gly3 residue. Energetically favourable rotamers of Tyr and Phc side chains were also determined for the μ‐ and δ‐conformations. The present models of μ‐ and δ‐conformations share geometrical similarity with the low‐energy structures of Leu‐enkephalin and the analogue.
Amidation of growth hormone releasing factor (1–29) by serine carboxypeptidase catalysed transpeptidation
Authors: BREDDAM KLAUS , WIDMER FRED , MELDAL MORTEN
Published: August 2010

The applicability of serine carboxypeptidase catalysed transpeptidation reactions, using amino acid amides as nucleophiles, for C‐terminal amidation of peptides has been investigated. With the aim of converting an unamidated precursor into GRF(1–29)‐NH2, an interesting biologically active derivative of growth hormone releasing factor, a number of model reactions were initially investigated. In such a transpeptidation reaction, where the C‐terminal amino acid is replaced by the amino acid amide, used as nucleophile, the C‐terminal amino acid residue of the substrate can be chosen freely since it functions as leaving group and does not constitute part of the product. Since the C‐terminal sequence of GRF(1–29)‐NH2 is ‐Met‐Ser‐Arg‐NH2 the model reactions Bz‐Met‐Ser‐X‐OH (X = Ala, Leu, Arg) + H‐Arg‐NH2→ Bz‐Met‐Ser‐Arg‐NH2+ H‐X‐OH were first studied. With carboxypeptidase Y and X = Ala or Leu the amidated product could be obtained of 98% and 41%, respectively. With carboxypeptidase W‐II and X = Arg a yield of no more than 72 % could be obtained. The choice of Ala as leaving group in combination with carboxypeptidase Y therefore appeared optimal. With the longer peptide Bz‐Leu‐Gln‐Asp‐Ile‐Met‐Ser‐Ala‐OH the amidated product could be obtained in a yield of 78%, using carboxypeptidase Y, the only other product being Bz‐Leu‐Gln‐Asp‐Ile‐Met‐Ser‐OH, formed due to the competing hydrolysis reaction. The full length peptide GRF(1–28)‐Ala‐OH was synthesized by the continuous flow polyamide solid‐phase method. With this peptide the yield of amidation was 87 % and the remaining products were GRF (1–28)‐OH and GRF(1–28)‐Arg‐Arg‐NH2. However, these can be separated from the desired product by ion exchange chromatography. The results demonstrate that it is feasible to use the generally stable and easily accessible serine carboxy‐peptidase for C‐terminal amidation of peptides, e.g. produced by fermentation of genetically manipulated microorganisms.
Structure and conformation of peptides containing the sulphonamide junction
Authors: CALCAGNI A. , GAVUZZO E. , LUCENTE G. , MAZZA F. , PINNEN F. , POCHETTI G. , ROSSI D.
Published: August 2010

The insertion of the (S)‐lactyl residue into the cyclodipeptide cyclo (‐Tau‐Pro‐) 3 leads in good yields to the first example of a stable tetrahedral adduct (oxa‐cyclol) 5 containing the sulphonamide junction. Compound 5 does not show a significant tendency towards tautomeric equilibria and possesses an unexpected syn‐orientation involving the hydroxyl group and the Pro‐Hα. The crystal structure and molecular conformation of 5 has been determined. Crystals are orthorhombic, s.g. P212121, with a = 6.607, b = 12.297, c = 16.622 Å. The cisoidal conformation around the S‐N bond is very similar to that found in the previously studied linear and cyclic peptides containing a sulphonamide junction. The taurine nitrogen is practically planar whereas the proline nitrogen, bound to the SO2 group, is highly pyramidal. In the tricyclic system of 5 the seven‐membered ring adopts a twist‐chair conformation while the pyrrolidine and oxazolidinone rings show an envelope conformation. The crystal packing is characterized by three hydrogen bonds all formed by means of a water molecule.
NMR studies on the conformation of cyclodipeptides with two identical L‐aromatic amino acid residues in solutions ‐ cyclo[‐l‐5(OH)Trp‐l‐5(OH)Trp] and cyclo[‐l‐Phe‐l‐Phe]
Authors: SHEINBLATT M.
Published: August 2010

NMR studies on the conformations of cyclodipeptides containing two identical l‐aromatic amino acid residues cyclo[‐l‐5(OH)Trp‐l‐5(OH)Trp] 8 and cyclo[‐l‐Phe‐l‐Phe] 9 in DMSO solutions are reported here. The 1H chemical shifts, the spin‐spin coupling constants, and the NOE results show that in these two cyclodipeptides one residue occupies the folded antiperpendicular and the second the extended to nitrogen perpendicular conformers respectively. The results indicate that each of the identical residues is in a fast conformational equilibrium between the above two conformers so that one set of 1H and 13C resonances is observed for the two identical residues in the above two cyclodipeptides.
A new detector for fully automatic peptide synthesis
Authors: FOX JOHN E. , NEWTON RAYMOND , STROUD CLAIRE H.
Published: August 2010

A new method of monitoring the rate of reactions in solid phase peptide synthesis is described. A conductivity detector in the reaction cell enables the deprotection, washing, and subsequent coupling stages to be examined in detail. The half lives of the reactions can be calculated and hence the optimum reaction times predicted. The aggregation of peptide chains and subsequent collapse of the resin is observed. Difficult sequences are sensed and appropriate action taken completely automatically.
Hydrolysis of oligoribonucleotides by α‐helical basic peptides
Authors: PERELLO MARGARITA , BARBIER BERNARD , BRACK ANDRÉ
Published: August 2010

Poly(Leu‐Lys‐Lys‐Leu) increases markedly the rate of hydrolysis of oligoribonucleotides. The polypeptide adopts an α‐helical conformation in water in the presence of salt. Non‐helical poly(Pro‐Lys‐Lys‐Leu) is much less active. Ac‐Leu‐Lys‐Lys‐Leu‐NHEt has no hydrolytic activity. Oligotetrapeptides Ac‐(Leu‐Lys‐Lys‐Leu)n‐NHEt with increasing chain‐length have been prepared by solid phase synthesis to evaluate the critical chain‐length required for the hydrolytic activity. It is possible to correlate the activity to the propensity to form α‐helices.
Synthesis and biological activity of 2‐phenylethyl ester analogues of C‐terminal heptapeptide of cholecystokinin modified in Trp 30 region
Authors: ROLLAND MARC , RODRIGUEZ MARC , LIGNON MARIE‐FRANÇOISE , GALAS MARIE‐CHRISTINE , LAUR JEANINE , AUMELAS ANDRÉ , MARTINEZ JEAN
Published: August 2010

We have tried to evaluate the significance of the tryptophan side chain residue and of the surrounding peptide bonds in the antagonist activity of cholecystokinin analogues lacking the C‐terminal amide function and having a d‐tryptophan. In order to perform this study, analogues of the C‐terminal heptapeptide of cholecystokinin were synthesized by replacing the C‐terminal phenylalanine residue with 2‐phenylethyl alcohol and by either replacing the tryptophan residue with an alanine, a norleucine and a phenylalanine residue, or introducing a “reduced peptide bond” in the tryptophan 30 region. Most of these compounds were able to reproduce only part of the response of cholecystokinin in stimulating amylase release from rat pancreatic acini, as was already observed for 2‐phenylethyl ester analogues of CCK. These results point out the key role of tryptophan 30 in the biological response of cholecystokinin.
Stabilities of leucine zipper dimers estimated by an empirical free energy method
Authors: KRYSTEK STANLEY R. , BRUCCOLERI ROBERT E. , NOVOTNY JIRI
Published: August 2010

The leucine zipper motif is a characteristic amino acid sequence found in dimeric DNA‐binding proteins. Computer‐generated models for leucine zippers were constructed as α‐helical coiled dimers with leucine repeated every seventh residue. An empirical Gibbs free energy, ΔG, function which incorporates hydrophobic force, electrostatic interactions, and conformational entropy loss as the major intermolecular interactions was used to estimate the ΔG of dimer formation in fos, jun, and GCN4 zipper sequences. The calculations showed that complexes known to form stable homo‐ or heterodimers have favorable (negative) ΔG, while other less stable complexes have unfavorable (positive) ΔG. Leucines in position d of the coiled coil contribute large hydrophobic stabilization energies while residues in the a position contribute less to dimer stability. Hydrophobic contributions show little sequence specificity, however, and do not contribute significantly to homo/heterodimer preference. Charged residues in the e and g positions, on the other hand, determine homo/heterodimer specificity. In GCN4 homodimers, residues GLU e1, Glu b2, Lys g2, and Lys e4 greatly contribute to dimer stability. The preferential stability of fos‐jun heterodimer over the jun‐jun and fos‐fos homodimers is primarily due to the side chains Asp b1, Glu g1, Asp b2, Glu e2, Glu g2, Glu g3, and Lys a5 of the fos helix, and Arg c1, Lys g1, Lys b2, Lys e2, Arg e4, and Glu g4 of the jun helix.
Purification of synthetic peptides Immobilized metal ion affinity chromatography (IMAC)*
Authors: LINDEBERG G. , BENNICH H. , ENGSTRÖM Å.
Published: August 2010

Immobilized metal ion affinity chromatography (IMAC) is a useful method for purification of synthetic peptides with an N‐terminal metal‐binding amino acid such as His, Trp, or Cys, especially when such residues are not present in other parts of the molecule. In solid phase peptide synthesis (SPPS), capping with acetic anhydride will, in principle, produce truncated peptides as the only side‐products due to incomplete couplings. Consequently, only the desired product will carry the affinity label. Most of the impurities, therefore, can be removed by a single passage through an IMAC column. Some representative examples are presented, where fairly large peptides (30–40 amino acid residues) were efficiently purified by this approach.
Linear and cyclic N‐terminal galanin fragments and analogs as ligands at the hypothalamic galanin receptor
Authors: LAND TUT , LANGEL OLO , LÖW MIKLOS , BERTHOLD MALIN , UNDÉN ANDERS , BARTFAI TAMAS
Published: August 2010

The neuropeptide galanin (1–29) binds with high affinity to hypothalamic receptors (KD∼ 0.9 nM) and regulates feeding behavior. The N‐terminal fragments (1–16), (1–16)NH2 are high affinity (KD∼ 6nM) full agonists in vivo and in vitro.l‐Ala substitutions show that amino acid residues Gly1, Trp2, Asn5, Tyr9, and Gly12 are important for the high affinity binding of galanin (1–16). Shortening the fragment (1–16) to galanin (1–7) causes a gradual drop of affinity: galanin (1–15), (1–14), and (1–13) have submicromolar KD values and galanin (1–12) has KD∼ 3 μm. Cyclic analogs of galanin (1–12) of different ring size were synthesized by condensing Gly1 and Gly12 without or with spacer groups. These analogs, independent of ring size, had a lower affinity than the linear galanin (1–12). Derivatization of the N‐terminus of galanin (1–29), (1–16), and (1–12) all resulted in a large drop of affinity for the receptors, suggesting again the importance of the free N‐terminal Gly.
Synthesis of polyamide supports for use in peptide synthesis and as peptide‐resin conjugates for antibody production
Authors: KANDA PATRICK , KENNEDY RONALD C. , SPARROW JAMES T.
Published: August 2010

We have synthesized beaded, hydrophilic cross‐linked, aminoalkyl polydimethylacrylamide supports upon which peptides have been assembled using standard Boc or Fmoc chemistry in automated equipment. The resins were prepared by the free radical‐initiated co‐polymerization of N,N‐dimethylacrylamide, N,N′‐bisacrylyl‐1,3‐diaminopropane, and a functional monomer which were contained in a reverse‐phase, detergent‐emulsified suspension. The functional monomers used were N‐(2‐(methylsulfonyl)ethyloxycarbonyl)‐allyl‐amine (MSC‐allylamine), N‐acrylyl‐1,6‐diaminohexane hydrochloride or N‐methacrylyl‐1,3‐diaminopropane hydrochloride. The MSC protecting group was removed by treatment of the resin with methanolic base during workup. After coupling of N‐α‐t‐butyloxycarbonyl‐alanine (Boc‐alanine), amino acid analyses gave resin loading capacities between 0.15 mmol/g and 1.4 mmol/g, depending on the concentration and composition of the functional monomer. The resulting polymers were highly swollen by polar solvents including aqueous buffers. Peptides were synthesized on these supports after attaching the first amino acid directly or through a cleavable ester linker. When the carboxyl‐terminal amino acid was coupled as the 4‐oxymethylbenzoic acid derivative, the peptide could be deprotected and remain attached to the hydrophilic polymer since the peptide‐benzyl ester bond was stable to HF deprotection at 0° in the presence of 10% anisole and 1% ethanedithiol. The resulting peptidyl‐resin could be swollen in aqueous buffers and injected into animals for the production of antibodies.
Investigation of cis/trans proline isomerism in a multiply occurring peptide fragment from human salivary proline‐rich glycoprotein
Authors: LOOMIS RONALD E. , GONZALEZ MIREYA , LOOMIS PAMELA M.
Published: August 2010

The solution‐state conformations of eight proline‐containing peptide fragments found in human salivary proline‐rich glycoprotein (PRG) were investigated in 2 × distilled water (treated with metal ion chelating resin) using 13C‐nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. The peptide sequences and acronyms were as follows: PRG9‐2 = NH2‐G(I)‐P(2)‐CONH2, PRG9‐3 = NH2‐G(1)‐P(2)‐P(3)‐CONH2,PRG9‐4 = NH2‐G(1)‐P(2)‐P(3)‐P(4)‐CONH2, PRG9‐5 = NH2‐G(1)‐P(2)‐P(3)‐P(4)‐H(5)‐CONH2,PRG9‐6 = NH2‐G(1)‐P(2)‐P(3)‐P(4)‐H(5)‐P(6)‐CONH2, PRG9‐7 = NH2‐G(1)‐P(2)‐P(3)‐P(4)‐H(5)‐P(6)‐G(7)‐CONH2, PRG9‐8 = NH2‐G(1)‐P(2)‐P(3)‐P(4)‐H(5)‐P(6)‐G(7)‐K(8)‐CONH2 and PRG9‐9 = NH2‐G(1)‐P(2)‐P(3)‐P(4)‐H(5)‐P(6)‐G(7)‐K(8)‐P(9)‐CONH2. Sequence‐specific resonance assignments from the 13C‐NMR spectra indicated that the trans proline isomer dominated the conformations of the peptides. CD results clearly showed the presence of the poly‐l‐proline II helix as the major conformation in PRG9‐3 → PRG9‐5, supplemented by β‐ and/or γ‐turns in PRG9‐6 → PRG9‐9. These data suggest that in “metal free” water, native PRG could contain several small poly‐l‐proline II helices along with β‐ and/or γ‐turns. Since proline is the major amino acid present in native PRG, these localized conformations may contribute to PRG's global conformation and act as a primary force in determining its biological activities.
Conformational prediction studies on pancreatic colipase
Authors: BELLON BERNARD , DEZAN CHRISTINE , RUGANI NATHALIE , SARDA LOUIS
Published: August 2010

Comparison of the primary structures of pancreatic colipases from man, pig, horse and rat shows a high degree of homology between proteins. Fifty‐two out of the 95 residues of the polypeptide are identical. All colipases contain 10 half‐cystines which are located at invariant positions. The secondary structure of colipases has been predicted from the sequence using the statistical method of Chou and Fasman and the method of Gibrat, Gamier and Robson based on information theory. Predictions indicate that colipases have a low content of α‐helix and β‐strand structure. The two segments at positions 7–10 and 56–59. assumed to be part of the lipid binding domain, have predicted β‐sheet conformation and should be in close spatial vicinity to each other in the proteins. Four β‐turns are predicted in all colipases at positions 3–6, 46–49, 61–64, and 81–84. They might contribute, with the five disulfide bridges, to a tight packing of the protein molecule. Surface residues and major sequential antigenic determinants of mammalian colipases have been predicted using methods based either on hydrophilicity/hydropathy scales or amino acid mutability. From these studies, it appears that colipases exhibit large conformational homologies. In the absence of data on the tertiary structure of colipase, predictive methods, together with physico‐chemical and immunological studies, provide valuable information on the conformation of the protein in relation to the topology of residues involved in the functional and antigenic sites.
A CD strategy for the study of polypeptide folding/unfolding
Authors: SILIGARDI GIULIANO , DRAKE ALEX F. , MASCAGNI PAOLO , ROWLANDS DAVID J. , BROWN FRED , GIBBONS WILLIAM A.
Published: August 2010

The circular dichroism spectrum of the 20‐residue immunogenic peptide from the foot‐and‐mouth disease virus (VP1; 141‐160 of serotype A, subtype 12) was solvent‐ and temperature‐dependent. Careful solvent titration revealed two isodichroic points and plateaux consistent with stepwise unfolding of specific stable conformations. Variable temperature studies in cryogenic solvents and urea perturbation were consistent with the existence of three conformational moieties, the left‐handed extended helix, the a‐helix, and the α‐helix. The number of residues in each helix was confirmed by CD spectral simulations. The strategy described here can be used to determine the components of a conformational equilibrium and their statistical weights, to study peptide folding and unfolding and to determine the bioactive conformation(s) of linear peptides. The conclusions were supported by 2D‐NMR studies. A new mechanism for the stabilization of left‐handed extended helices and destabilization of a‐helices by urea is proposed. The structure of the peptide as resolved by CD spectroscopy is of particular significance since the conformation of this antigenic sequence in situ has so far not been solved by X‐ray crystallography.
Application of 2‐chlorotrityl resin in solid phase synthesis of (Leu15)‐gastrin I and unsulfated cholecystokinin octapeptide
Authors: BARLOS KLEOMENIS , GATOS DIMITRIOS , KAPOLOS STAVROS , POULOS CONSTANTINE , SCHÄFER WOLFRAM , WENQING YAO
Published: August 2010

The carboxyl terminal dipeptide amide, Fmoc‐Asp‐Phe‐NH2, of gastrin and cholecystokinin (CCK) has been attached in high yield through its free side chain carboxyl group to the acid labile 2‐chlorotrityl resin. The obtained peptide resin ester has been applied in the solid phase synthesis of partially protected (Leu15)‐gastrin I utilising Fmoc‐amino acids. Quantitative cleavage of this peptide from resin, with the t‐butyl type side chain protection intact is achieved using mixtures of acetic acid/trifluoroethanol/dichloro‐methane. Under the same conditions complete detritylation of the tyrosine phenoxy function occurs simultaneously. Thus, the solid‐phase synthesis of peptides selectively deprotected at the side chain of tyrosine is rendered possible by the use of 2‐chlorotrityl resin and Fmoc‐Tyr(Trt)‐OH. The efficiency of this approach has been proved by the subsequent high‐yield synthesis of three model peptides and the CCK‐octapeptide.
Biological effects and receptor binding affinities of new pseudononapeptide bombesin/GRP receptor antagonists with N‐terminal d‐Trp or d‐Tpi
Authors: RADULOVIC SINISA , CAI REN‐ZHI , SERFOZO PETER , GROOT KATE , REDDING TOMMIE W. , PINSKI JACEK , SCHALLY ANDREW V.
Published: August 2010

In an attempt to produce more powerful (effective) bombesin/GRP receptor antagonists, the d forms of Trp or Trp analog (Tpi) were introduced at position 6 in two pseudononapeptides, Leu13Ψ (CH2NH)Leu14‐bombesin(6‐14) and Leu13Ψ(CH2NH)Phe14 ‐bombesin (6‐14). These antagonists were tested for their ability to inhibit basal and gastrin releasing peptide (GRP) (14‐27)‐induced amylase release from rat pancreatic acini in a superfusion assay. They were also assessed for the inhibition of 125I‐Tyr4 ‐bombesin binding to Swiss 3T3 and small cell lung carcinoma cell line H‐345 and the mitogenic response of Swiss 3T3 cells induced by GRP(14‐27). The peptides, when given alone, did not stimulate amylase secretion, but were able to inhibit gastrin releasing peptide (14‐27)‐induced amylase release. All of the antagonists showed strong binding affinities for Swiss 3T3 and H‐345 cells and suppressed the GRP(14‐27)‐induced increase of [3H]thymidine incorporation into DNA of Swiss 3T3 cells at nanomolar concentrations. Antagonist d‐Tpi6,Leu13Ψ (CH2NH)Leu14‐bombesin (6‐14)(RC‐3095) was slightly more potent in these assays than d‐Trp6,Leu13Ψ (CH2NH)Leu14‐bombesin (6‐14)(RC‐3125). Nevertheless, d‐Trp6 Leu13Ψ (CH2NH)Phe14‐bombesin (6‐14) showed the highest binding affinity for Swiss 3T3 and H345 cells and it was the most potent inhibitor of GRP(14‐27)‐induced amylase secretion. This antagonist RC‐3420 was particularly effective in inhibiting the growth of Swiss 3T3 cells, exhibiting an IC50 value less than 1 nm. Our work indicated that the substitution of d‐Trp and d‐Tpi at position 6 of the pseudononapeptide bombesin analogs (Ψ13‐14), in which the Met14 residue is replaced by Leu or Phe, results in potent bombesin/GRP antagonists with improved in vivo activity.
Evidence for a β‐turn in an azadipeptide sequence
Authors: BENATALAH ZOHRA , AUBRY ANDRÉ , BOUSSARD GUY , MARRAUD MICHEL
Published: August 2010

Crystalline ButCO‐Pro‐AzaAla‐NHPri, prepared by the active ester procedure, has been subjected to single‐crystal X‐ray diffraction analysis which evidences a type II β‐turn.
Effect of copper(II) chloride on suppression of racemization in peptide synthesis by the mixed anhydride and related methods
Authors: MIYAZAWA TOSHIFUMI , DONKAI TOMOKO , YAMADA TAKASHI , KUWATA SHIGERU
Published: August 2010

In segment couplings by the mixed anhydride method using isobutyloxycarbonyl chloride, the use of copper(II) chloride as an additive suppressed racemization completely in the same manner as in the carbodiimide method reported previously. This was confirmed by employing a number of couplings between Z‐dipeptides and amino acid esters. The racemization‐suppressing effect of other compounds were also evaluated by employing one of these model couplings to be at best only limitedly effective. Copper(II) chloride was effective also in the related method using EEDQ. Thus, in the couplings where a low level of racemization was observed without an additive, the addition of copper(II) chloride eliminated racemization even at ambient temperature where EEDQ is usually used. The effectiveness of copper(II) chloride was confirmed also in the BOP‐C1 method. In the presence of HOBt racemization was reduced to a low but still detectable level, while it was suppressed completely by the addition of copper(II) chloride.
Structural requirements for biological activity of glucagon‐like peptide‐I
Authors: MOJSOV SVETLANA
Published: August 2010

Glucagon‐like peptide‐I (GLP‐I) is encoded together with glucagon by the glucagon gene and is related in its structure to the glucagon‐secretin family of peptides. Three of the predicted forms of the peptide, a 37‐residue long GLP‐I(1–37), a 31‐residue GLP‐I(7–37) and a 30‐residue GLP‐I(7–36) amide as well as three analogs des [Gly37, Arg36] GLP‐I(7–37), des [Gly37, Arg36, Gly35] GLP‐I(7–37) and des [His7] GLP‐I(7–37) were synthesized by the stepwise solid phase method. These synthetic peptides were used to define the structural domains required for the binding of GLP‐I to the pancreatic beta cell. The competitive binding experiments showed that both the amino and carboxyl terminal domains of the molecule contribute to GLP‐I binding. In these experiments glucagon, another peptide that stimulates insulin secretion, was a weak full agonist of GLP‐I binding. Results from these studies provide further characterization of the physiological role of this new peptide.
Purification of synthetic peptides using reversible chromatographic probes based on the Fmoc molecule
Authors: BALL H.L. , MASCAGNI P.
Published: August 2010

A rapid, versatile, reversible procedure for purifying synthetic peptides has been developed based on the specific incorporation of 4‐carboxylate Fmoc derivatives onto the terminal amino acid of peptidyl‐resins. The acid stable 4‐COR‐Fmoc derivatives were synthesised with a variety of chemical groups thus altering the chromatographic properties of the “target” peptides and permitting their convenient purification, either by reversed‐phase HPLC or ion exchange chromatography. The assembly of the peptides involved a capping step to prevent the formation of deletion forms. The 4‐COR‐Fmoc derivatives were incorporated either as preformed amino acid conjugates or as activated succinimidyl esters. After HF cleavage and purification the 4‐COR‐Fmoc probes were quantitatively removed with organic bases. The efficiency of the technique was demonstrated by the purification of small to large sized peptides, including a cyclic analogue.
A novel, mild, specific and indirect maleimido‐based radioiodolabeling method
Authors: CHOREV# MICHAEL , CAULFIELD MICHAEL P. , ROUBINI ELIAHU , MCKEE ROBERTA L. , GIBBONS SUSAN W. , LEU CHIH‐TAI , LEVY JAY J. , ROSENBLATT MICHAEL
Published: August 2010

In an effort to design a mild, non‐oxidative and site‐specific means of radiolabeling bioactive molecules we have employed maleimido‐sulfhydryl chemistry to produce bioactive hormone radioligands. We have prepared two novel radioiodolabeled reagents, 3′‐maleimidopropanoyl‐3‐125I‐tyramide and its retro analog, N‐maleoyl‐N′‐3‐(4‐hydroxy‐3‐125I‐phenyl)propanoy1 ethylenediamide, by either oxidative radioiodination of the precursors or radiolabeling of the phenolic component prior to its incorporation into the radiolabeling reagents. These reagents were then used to radiolabel analogs of parathyroid hormone (PTH) and parathyroid hormone‐related protein (PTHrP) in an efficient way, yielding reaction mixtures which were easily purified. The radioligands obtained are stable upon storage and bind in a reversible manner to a single population of binding sites displaying affinity in the low nanomolar range. The potencies of these analogs are comparable to the non‐modified PTH and PTHrP analogs. This study demonstrates the utility of the novel maleimido‐based indirect radioiodination approach and highlights some of its advantages over either direct oxidative procedures or acylation using the Bolton‐Hunter reagent.
Racemization studies in peptide synthesis through the separation of protected epimeric peptides by reversed‐phase high performance liquid chromatography
Authors: MIYAZAWA TOSHIFUMI , OTOMATSU TOSHIHIKO , YAMADA TAKASHI , KUWATA SHIGERU
Published: August 2010

Separation of protected epimeric peptides, Z‐Gly‐Xaa‐Xbb‐OMe (where Xaa and Xbb = chiral amino acid residues), by reversed‐phase HPLC was utilized for studying racemization in peptide synthesis. Thus, the following factors which might affect the extent of racemization during the coupling by the carbodiimide method were investigated: the combination of amino acid residues to be coupled, coexisting tertiary amine salts, and the relative configuration of the amino acid residues. The following points were revealed: the combination of bulky residues at the coupling site results in extensive racemization in a polar solvent such as DMF, the amine hydrochlorides cause less racemization than the p‐toluenesulfonates in DMF, and the influence of relative configuration differs depending on the solvent and the individuality of the amino components. Furthermore, the racemization‐suppressing effect of some additives in the carbodiimide method was reevaluated by employing the same procedure.
Crystal structure and molecular conformation of achatin‐I (H‐Gly‐d‐Phe‐Ala‐Asp‐OH), an endogenous neuropeptide containing a d‐amino acid residue
Authors: ISHIDA TOSHIMASA , IN YASUKO , DOI MITSUNOBU , INOUE MASATOSHI , YASUDA‐KAMATANI YOSIMI , MINAKATA HIROYUKI , IWASHITA‐ TAKASHI , NOMOTO KYOSUKE
Published: August 2010

In order to investigate the active conformation of achatin‐I (H‐Gly‐d‐Phe‐Ala‐Asp‐OH), an endogenous neuropeptide from the Achatina fulica ganglia, its crystal structure and molecular conformation were analysed by the X‐ray diffraction method. Crystals from methanol/dioxane are monoclinic, space group P21 with a=5.083(1), b= 9.125(1), c= 20.939(3) Å, β=94.73(1)° The structure was solved by direct methods and refined to R = 0.051 for 1714 independent reflections with Fo > σ(Fo). The molecule exists as a zwitterion with the Gly N‐terminal end protonated and Asp β‐carboxyl deprotonated; the C‐terminal of Asp is in a neutral state. The molecule takes a kind of β turn structure with the d‐Phe‐Ala residues at the corner of the bend. This turn conformation is primarily formed by the strong intramolecular hydrogen bonds of NH(Gly)—Oδ1 (Asp) and NH(Asp)‐ Oδ1(Asp) pairs, thus forming a 15‐membered ring structure. Judging from the published data concerning the structure‐activity relationship, this turn conformation may reflect an important feature related to the neuroexcitatory activity of achatin‐I.
Interaction of glucagon with artificial lipid bilayer membranes
Authors: KIMURA SHUNSAKU , ERNE DANIEL , SCHWYZER ROBERT
Published: August 2010

The enhancement of fluorescence emission from the tryptophan residue of glucagon, the quenching of that emission with acrylamide and with 5‐doxyl and 16‐doxyl stearic acid, circular dichroism spectra, the release of 6‐carboxytluorescein, and polarized infrared attenuated total reflection (IR‐ATR) spectra were used to study the interaction of glucagon with intact lipid vesicles and flat bilayers. Dimyristoylphosphatidylcholine bound the peptide only below the main transition temperature, thus confirming earlier results of Epand et al. (1977). However, the peptide is also bound by vesicles of unsaturated lipids above their transition temperature, suggesting an influence of lipid area on the binding process. Circular dichroism showed that binding to such vesicles also increases the helix content of glucagon. The IR‐ATR study and a comparison with dynorphin‐A‐(I‐13)‐tridecapeptide revealed profound differences in orientation of the two peptides. The dichroic ratios and the derived order parameters indicated an isotropic orientation of the helical segments of glucagon, but did not exclude a principal orientation of the molecules lying flat on the nienibrane surface. In contrast, the axis of the dynorphin helix is clearly oriented normal to the interface. The two peptides also differ in their rates of 6‐carboxyfluorescein release, suggesting a deeper penetration of the primary amphiphilic helix of dynorphin A‐(I‐13) than of the secondary amphiphilic helix of glucagon.
Effect of alkylation with different sized substituents on thermal stability of lysozyme
Authors: FUJITA YUKIHISA , NODA YUKINAO
Published: August 2010

The amino groups of hen egg white lysozyme were reductively alkylated by the reaction with aliphatic aldehydes of various chain lengths and with two aldehydes of different steric hindrance at pH 7.5 and 4° for 3 h. About four of the original six lysine residues were modified by the reaction with acetaldehyde, n‐butylaldehyde or n‐hexylaldehyde. About three lysine residues were 2, 2‐dimethylpropylated with trimethy‐lacetaldehyde while a single residue was modified with benzaldehyde. The thermal stabilities of these alkylated lysozymes were investigated by differential scanning calorimetry (DSC) at different acidic pH values. Alkylation thermally destabilized the proteins, depending not only on the extent of modification but also on the size of the substituent. The alkylated derivatives were 8–19kJ/mol less stable than native lysozyme at 25° and pH 3.0. The temperature dependences of the activities of the alkylated lysozymes against ethylene glycol chitin indicated that the orders of the optimum temperatures and the maximum activities were exactly the same as the order of the thermal stabilities.
High level synthesis of biologically active recombinant trichosanthin in Escherichia coli
Authors: ZHU RONG‐HUAN , NG TZI‐BUN , YEUNG HIN‐WING , SHAW PANG‐CHUI
Published: August 2010

Two forms of recombinant trichosanthin (rTCS) were synthesized in high levels in Escherichia coli by putting the TCS cDNA under the control of a T7 RNA polymerase‐directed promoter. Purification schemes were developed to isolate the recombinant protein from both soluble and insoluble fractions. Form I rTCS possessed the mature TCS sequence and had similar biological activities as the natural protein. Its IC50 was approximately 0.13 nm in an in vitro rabbit reticulocyte translational system and a dose of around 35 μg protein per 25 g body weight was sufficient to induce complete abortion in mice. Form II rTCS had a propeptide of 19 aa at the C‐terminus and was five times less active than Form I in inhibiting protein synthesis by a rabbit reticulocyte lysate.
Preparation of protected peptide amides using the Fmoc chemical protocol
Authors: STORY SANDRA C. , ALDRICH JANE V.
Published: August 2010

Different resins were examined for their potential use in the solid phase synthesis of protected peptide amides using the 9‐fluorenylmethoxycarbonyl (Fmoc) chemical protocol. The model protected peptide amide BocTyr‐Gly‐Gly‐Phe‐Leu‐Arg(Pmc)NH2 (1) was synthesized on both the acid‐labile 4‐(2′,4’‐dimethoxyphenyl‐Fmoc‐aminomethyl)phenoxy resin (Rink amide resin) (2) and on resins containing the base‐labile linker 4‐hydroxymethylbenzoic acid. Of the resins examined only the methylbenzhydrylamine resin containing the 4‐hydroxymethylbenzoic acid linkage, which was cleaved by ammonolysis in isopropanoll, gave the model peptide 1 in good overall yield (53% including functionalization). Thus the synthesis of protected peptide amides by solid phase synthesis using Fmoc‐protected amino acids with t‐butyl‐type side chain protecting groups is feasible. The choice of peptide‐resin linkage and its cleavage conditions, however, are critical to the success of such syntheses. The potential application of this synthetic strategy to the preparation of novel peptide amides is discussed.
Evaluation of biological activities of new LH‐RH antagonists (T‐series) in male and female rats
Authors: PINSKI JACEK , YANO TETSU , JANAKY TAMAS , NAGY ATTILA , JUHASZ ATTILA , BOKSER LAZARO , GROOT KATE , SCHALLY ANDREW V.
Published: August 2010

A series of new highly potent LH‐RH antagonists (T‐series) has been synthesized in our laboratory. Among these analogs, antagonists [Ac‐d‐Nal(2), d‐Phe(4Cl)2. d‐Pal(3)3, d‐Lys(A2pr(Car)2)6, d‐Ala10LH‐RH (T‐140); [Ac‐d‐Nal(2)1, d‐Phe(4Cl)2, d‐Pal(3)3, d‐Lys(A2pr(Ac)2)6, d‐Ala10]LH‐RH (T‐148); [Ac‐d‐Nal(2)1, d‐Phe(4Cl)2, d‐Pal(3)3, d‐Lys(A2pr(For)2)6, d‐Ala10)]LH‐RH (T‐151) and [Ac‐d‐Nal(2)1, d‐Phe(4cl)2, d‐Pal(3)3, d‐Lys(A2bu(For)2)6, d‐Ala10]LH‐RH (T‐159) were the most powerful. Antagonists T‐140, T‐148 and T‐151 produced a complete blockade of ovulation in normal cycling rats at a dose of 1.5 μg/rat and antagonist T‐159 at a dose of only 0.75 μg/rat. The inhibitory effects of compounds T‐148, T‐151 and T‐159 on gonadotropin and sex steroid secretion were investigated in male and female rats. To determine their effect on LH levels in castrated male and ovariectomized female rats, T‐148, T‐151 and T‐159 were injected subcutaneously in doses of 0.625 and 2.5 μg/rat. Blood samples were taken at different intervals for 48 h. All three compounds at either dose caused a significant (P< 0.01) decrease in LH levels for more than 6 h. Significant (P <0.01) inhibition of LH lasted for more than 24 h following a dose of 2.5 μg sc of all 3 antagonists in both male and female rats. Serum FSH levels were also suppressed significantly for more than 48 h in castrated male rats by all three antagonists at a dose of 5 μg/rat sc. Serum testosterone levels were measured in intact male rates injected with antagonists T‐148, T‐151 and T‐159 in doses of 50 and 100 pg sc. Both doses produced a dramatic fall in testosterone (P<0.01) to castration levels 6 h after injection. The inhibition of serum testosterone lasted for more than 48 h, but only 100 μg of T‐148 maintained testosterone in the castration range for more than 48 h. Antagonists T‐148, T‐151 and T‐159 injected at a dose of 100 μg to intact female rats reduced serum estradiol levels significantly (P<0.01) for more than 48 h, as compared to control animals. In the cutaneous anaphylactoid test (CAT), T‐148, T‐151 and T‐159 proved to have very low histamine releasing activities. These data demonstrate a high efficacy of these new LH‐RH antagonists in suppressing the pituitary‐gonadal axis in male and female rats. These LH‐RH antagonists could possibly be used for treatment of sex‐hormone sensitive cancers and other disorders and conditions, in which a reduction in circulating sex steroids would be beneficial.