bluuebunny/crossref_metadata_2025 huggingface dataset:
Association of β‐agonists with corresponding β2‐ and β1‐adrenergic pentapeptide sequences
Authors: SCHMIDT WALTER F. , WATERS ROLLAND M. , MITCHELL ALVA D. , WARTHEN J.D. , HONIGBERG IRWIN L. , VAN HALBEEK HERMAN
Published: August 2010

Synthesized β1‐ and β2‐pentapeptide sequences corresponding to published adrenoceptor transmembrane activation site subtypes were investigated in vitro for selectivity in association for drug ligands of known selectivity. Both nuclear magnetic resonance spectroscopy and molecular mechanics demonstrated that structural differences among the corresponding pentapeptide activation‐site sequences can explain agonist selectivity. Results suggest the agonists bind across the activation site loop on the second transmembrane α‐helix by dipole/dipole interactions between a ligand and the peptide. Since electrostatic interactions within the membrane may determine the rate of intercellular ion flux, agonist association across the activation site sequence could thereby decrease electrostatic resistance to positive ion flux into the cell. Interactions between the peptides and the ligands may provide insight into the structures and mechanisms involved in association of ligands for the identical sequences on the β‐adrenoreceptors.
Interleukin‐1β converting enzyme
Authors: REITER LAWRENCE A. , MARTIN JOHN J.
Published: August 2010

A series of compounds containing a hydroxyethyl‐based dipeptide surrogate have been prepared as probes to evaluate the possibility of ICE being an aspartic protease. The aldehyde t‐BocAsp(β‐t‐butyl)H reacted with the organochromium species derived from phenethyl bromide and CrCl2 to give the expected addition product. Lactonization, reprotection of the amine and oxidation with RuCl3 gave the two protected dipeptide surrogates 7a and 7b. These were incorporated into tetra‐, penta‐ and hexapeptide‐like molecules and evaluated as inhibitors of the enzyme. The failure of these compounds to inhibit ICE indicated that this enzyme was very unlikely to be an aspartic protease.
Solid‐phase synthesis of [Ala1,3,11,15]‐endothelin‐1 by a modified double‐coupling procedure
Authors: GREEN JEREMY
Published: August 2010

[Ala1,3,11,15]‐Endothelin‐1, a linear analogue of endothelin‐1 in which alanines replace the cystine residues, has been prepared by solid‐phase synthesis in approximately 17% yield. Fmoc amino acids were coupled using an economical modified double‐coupling (symmetrical anhydride followed by O‐benzotriazolyl ester) procedure under fully automated conditions. Conditions for synthesis and purification were closely monitored and should be applicable to other endothelin analogues.
Design of helix ends
Authors: DASGUPTA SWAGATA , BELL JEFFREY A.
Published: August 2010

The amino acid sequence and chemical interactions at the ends of 163 helices were surveyed so as better to understand amino acid preferences previously observed [Richardson, J.S. & Richardson, D.C. (1988) Science240, 1648–1652]. Amino acid preferences differed from the previous survey in some significant details and in ways that might affect the choice of amino acids during the design of a protein helix. The following major conclusions about helix ends were deduced from additional patterns of amino acid occurrence and interactions that were observed. (1) A specific pair of hydrogen bonds is often observed between a glutamic acid (or glutamine) side chain at the N3 position and the N‐cap amide hydrogen, and between the N‐cap side chain (often threonine) and the N3 amide hydrogen. This reciprocal interaction may be an important means of stabilizing the N‐terminal end of a helix. (2) Negatively charged amino acids (aspartic acid and glutamic acid) at the N‐terminal end of helices may be more important in stabilizing protein helices than positively charged residues (chiefly lysine) at the C‐terminal end. (3) The identity of the residue at the N‐cap position is correlated with the backbone conformation at that position. (4) Aspartic acid (or asparagine) at the N2 or N3 position may adopt a conformation that suggests a hydrogen‐bonding interaction with the end of the helix, especially when the N‐cap side chain does not form a hydrogen bond with the end of the helix.
Synthesis and biological activity of a destruxin analogue: d‐Lac‐6 destruxin E
Authors: CALMES MONIQUE , CAVELIER‐FRONTIN FLORINE , JACQUIER ROBERT , MERCADIER JEAN‐LUC , SABIL SAMIA , VERDUCCI JEAN , QCIOT JEAN‐MARIE , VEY ALAIN
Published: August 2010

A general strategy for the synthesis of destruxin analogues is described and applied to a particular example, d‐Lac‐6 destruxin E. The tetrapeptide Boc‐Ile‐N‐MeVal‐N‐MeAla‐β‐Ala‐OMe (2) was chosen as the basic starting compound, and its preparation was optimized. This fragment was then coupled with the compound (D)Lac‐Pro, and the resulting peptide was cyclized by the DEPC or DPPA/HOBt/DMAP methods at 21 and 30% yield, respectively. The biological activity of the analogue obtained was established by injection to an insect host.
Synthesis of phosphotyrosine‐containing peptides by the solid‐phase method
Authors: TIAN ZHENPING , GU CHING , ROESKE ROGER W. , ZHOU MINGMING , VAN ETTEN ROBERT L.
Published: August 2010

A practical and convenient procedure for making phosphotyrosine‐containing peptides by the solid‐phase method was developed. Phosphotyrosine was incorporated via Boc‐Tyr(PO3Bzl2)‐OH. The completed peptide was cleaved from the solid support by treatment with 1 M TMSBr‐thioanisole‐TFA. By gel‐phase 31P‐NMR spectroscopy we found that one of the benzyl protecting groups on phosphate was completely removed by two consecutive runs of Boc deprotection with 50% TFA‐DCM. However, the other benzyl group remained intact throughout the synthesis (35 cycles).
Solvation of human serum albumin in aqueous alkylurea solutions
Authors: KRANJC ZDENKA , LAPANJE SAVO
Published: August 2010

Solvation of human serum albumin in aqueous solutions of urea, methyl‐, N,N′ ‐dimethyl‐, and ethylurea was studied by density measurements. From the densities at constant molality and constant chemical potential, the preferential solvation parameters of human serum albumin were determined. In all solutions the denaturant is preferentially bound. From preferential solvation data, Gibbs free energies of transfer from water to alkylurea solutions were calculated. Since the enthalpies of transfer were determined previously the entropies of transfer would also be obtained. Based on these and model compound data an attempt is made to identify various interactions involved in solvation. The total binding of denaturant to protein could also be calculated from preferential solvation data. The following major binding sites have been identified: ionic groups on the surface of protein molecules, peptide groups and aromatic side‐chains. The correlation between the total number of sites and the number of urea molecules bound is satisfactory.
Allyl‐based groups for side‐chain protection of amino‐acids
Authors: LOFFET A. , ZHANG H.X.
Published: August 2010

Allyl and allyloxycarbonyl groups are used for the side‐chain protection of amino acids. The protecting groups may be selectively cleaved using the reagent HSnBu3 under palladium catalysis. The preparation of Boc and Fmoc series of protected amino acids is described.
Cyclic hexapeptide analogs of somatostatin containing bridge modifications
Authors: HUANG ZIWEI , PRÖBSTL ALBERT , SPENCER JEFFREY R. , YAMAZAKI TOSHIMASA , GOODMAN MURRAY
Published: August 2010

The cyclic hexapeptide c[Pro6‐Phe7‐D‐Trp8‐Lys9‐Thr10‐Phe11] displays higher bioactivity than native somatostatin in inhibiting the release of growth hormone. The superscript numbers refer to the location of the residues in native somatostatin. To investigate the structural role played by the Phe11‐Pro6bridging region, we have synthesized a series of cyclic hexapeptide analogs of somatostatin incorporating peptidomimetics and retro‐inverso modifications at the bridging region. Among them, two analogs contain the retro‐inverso modification mAla6‐gPhe11at the bridging region, and five analogs contain 2‐aminocyclopentane carboxylic acid (2‐Ac5c) and 1‐aminocyclopentane carboxylic acid (1‐Ac2c) as proline mimetics. The conformational preferences of these analogs have been studied using1H‐NMR and computer simulations. All of these analogs maintain conformations similar to those of the parent cyclic hexapeptide around the Phe7‐D‐Trp8‐Lys9‐Thr10tetrapeptide region consisting of a β II′ turn. However, they display different conformational features around the bridging region. The R‐mAla analog and the fiveAc5canalogs show only atransamide bond for Phe11‐Pro6in the bridging region, while the S‐mAla analog displays acis transisomerization for the same amide linkage in the bridging region. TheR‐mAla and the fiveAc5canalogs do not bind to the somatostatin receptor, while the S‐mAla analog displays a high binding activity. Applying our recently proposed model for bioactivity of somatostatin analogs, we examined the structure‐bioactivity relationships for these somatostatin analogs. This investigation provides valuable insight into the structural role played by the bridging region.
First characterization at atomic resolution of the C‐activating groups in a peptide synthesis acid chloride, acid azide and carboxylic‐carboxylic mixed anhydride*
Authors: CRISMA M. , MORETTO V. , VALLE G. , FORMAGGIO F. , TONIOLO C.
Published: August 2010

The X‐ray diffraction structures of three Nα‐protected, Cα‐activated MeAib derivatives are reported. They are Tos‐MeAib‐Cl, Tos‐MeAib‐N3 and Tos‐MeAib‐O‐Piv. The geometry and conformation of these classical carboxyl activating groups, which have been characterized at atomic resolution for the first time, are discussed.
Structure‐activity and conformational studies of a series of modifiedC‐terminal hexapeptide neurotensin analogues
Authors: HEYL DEBORAH L. , SEFLER NDREA M. , HE JOHN X. , SAWYER TOMI K. , WUSTROW DAVID J. , AKUNNE HYACINTH C. , DAVIS M. DUFF , PUGSLEY THOMAS A. , HEFFNER THOMAS G. , CORBIN ANN E. , CODY WAYNE L.
Published: August 2010

Neurotensin (NT), is a linear tetradecapeptide (pGlu1‐Leu2‐Tyr3‐Glu4‐Asn5‐Lys6‐Pro7‐Arg8‐Arg9‐Pro10‐Tyr11‐Ile12‐Leu13) that has been found in the central nervous system and peripheral tissues and appears to have a variety of physiological properties. AC‐terminal hexapeptide analogue [Nx‐Arg‐Lys‐Pro‐Trp‐Tle‐Leu, (1) Tle =tert‐leucine] has recently been reported to have high affinity for the NT receptor and appears to possess central activity after systemic administration. In an effort to probe the structure‐activity and conformational properties of the dipeptide, Pro‐Trp for binding and functional activity, these residues have been substituted with several natural and unnatural amino acids. Some of these analogues have binding affinities similar to compound 1, while in other cases, such as D‐amino acid substitutions, the peptides had negligible binding affinity. In general, the Pro10position seems more tolerant of substitution by amino acids that favor a reverse turn, rather than those that favor an extended conformation. The Trp11position accepted extra steric bulk more readily than conformational constraints.
Inductive effects on the structure of proline residues
Authors: PANASIK NICHOLAS , EBERHARDT ERIC S. , EDISON ARTHUR S. , POWELL DOUGLAS R. , RAINES RONALD T.
Published: August 2010

4(S)‐Hydroxyproline (Hyp) residues constitute about l0%, of most forms of collagen, the most abundant protein in vertebrates. X‐Ray diffraction analysis was used to ascertain how the structure of proline residues is affected by the inductive effect elicited by the hydroxyl group of Hyp residues. N‐Acetylproline methylester (1), N‐acetyl‐4(S)‐hydroxyproline methylester (2) and N‐acetyl‐4(S)‐fluoroproline methylester (3) were synthesized, and their crystalline structures were determined at high resolution. The amide bond of crystalline 1 was in the cis conformation. which is the minor isomer in solution, and the pyrrolidine ring of 1 had Cγ‐endo pucker. In crystalline 2 and 3 the arnide bonds were in the trans conformation, and the pyrrolidine rings had Cγ‐exo pucker. The lengths of the bonds between sp3‐hybridized carbon atoms in the pyrrolidine ring were significantly shorter in 2 and 3 than in 1, as was predicted by ab initio molecular orbital calculations at the RHF/3‐21G level of theory. No significant change in bond length was observed in the other bonds of 1, 2 or 3. The pyramidylization of the nitrogen atom increased dramatically in the order: 1<2<3. Together, these results indicate that electron‐withdrawing substituents in the 4‐position of proline residues can have a significant influence on the structure of these residues. In particular, the change in pyramidylization suggests that such substituents increase the sp3‐character of the prolyl nitrogen atom and could thereby alter the rate of prolyl peptide bond isomerization.
Amino acid sequence and properties of vasopressin‐associated elephant neurophysin
Authors: HUANG HSIEN‐BIN , WELLNER DANIEL , NAUDE RYNO , OELOFSEN WILLEM , OOSTHUIZEN MATHYS M.J. , BRESLOW ESTHER
Published: August 2010

The primary structure of an elephant neurophysin, homologous to vasopressin‐associated neurophysins, is reported. The protein contains a Tyr for Asn substitution at position 75, a position in direct contact with residues 77 and 78 of the monomer‐monomer interface. This Tyr residue therefore serves as a potential reporter of the path involved in the long‐range linkage between peptide binding and dimerization in this system. NMR studies of the protein in unliganded and liganded states demonstrated normal dimerization properties and the expected increase in dimerization associated with binding peptide. In keeping with an elevated pKa of 11.1 assigned to Tgr‐75 by UV spectrophotometric titration, the NMR signals from the 3,5 and 2,6 ring protons of Tyr‐75 were shifted 0.3 and 0.2 ppm upfield, respectively, relative to their positions in small peptides, indicating significant shielding and for hydrogen bonding. The Tyr‐75 ring proton signals narrowed slightly, with no discernible change in chemical shift, on conversion from dimer to monomer in the unliganded state. Ring protons of Tyr‐49, distant from the monomer‐monomer interface, but adjacent to the peptide‐binding site, were markedly perturbed by dimerization, in accord with their behavior in bovine neurophysins. The results suggest that the secondary and tertiary structure of the region 75–78 is largely unchanged by dimerization, and argue against an important role for this region in dimerization‐mediated conformational changes that alter the binding site in the unliganded state. On the other hand, binding of peptide was associated with both measurable shifts and broadening of Tyr‐75 ring NMR signals, consistent with high‐pressure studies which have suggested ligand‐induced changes in the physical properties of the monomer‐monomer interface.
Structural and mechanistic implications of incorporating naturally occurring aberrant mutations of human dihydropteridine reductase into a rat model
Authors: VARUGHESE KOTTAYIL I. , XUONG NGUYEN‐H. , WHITELEY JOHN M.
Published: August 2010

Phenylketonuria (PKU) is a debilitating hereditary disorder related to an individual's inability to convert phenylalanine to its usual tyrosine product. The genetic errors occur in three regions: in the cooperative enzymes phenylalanine hydroxylase (PAH) and dihydropteridine reductase (DHPR), and in the biosynthetic pathway from GTP to the hydroxylation cofactor, tetrahydrobiopterin (BH4). Many instances of naturally occurring defects in DHPR metabolism have been identified, and in most cases the error has been equated with an altered enzyme gene sequence. Using computer graphics, this report analyses the altered structural characteristics of eight of the enzymes encoded by mutant gene sequence and provides logical explanations for their diminished enzyme activities. In one instance, that of a threonine insertion, a mutant construct of the rat analog has been expressed in Escherichia coli and the DHPR isolated and characterised, confirming the marked changes this insert can create.
Comparative conformational analyses of μ‐selective dermorphin and §‐selective deltorphin‐II in aqueous solution by1H‐NMR spectroscopy
Authors: SEGAWA MOTOZUMU , OHNO YASUNORI , DOI MITSUNOBU , INOUE MASATOSHI , ISHIDA TOSHIMASA , IWASHITA TAKASHI
Published: August 2010

Two‐dimensional1H‐NMR methods have been used to obtain complete proton resonance assignments and possible solution conformations of dermorphin (H‐Tyr‐D‐Ala‐Phe‐Gly‐Tyr‐Pro‐Ser‐NH2) and deltorphin‐II (H‐Tyr‐D‐Ala‐Phe‐Glu‐Val‐Val‐Gly‐NH2), naturally occurring μ and §‐selective opioids, respectively, in order to examine the conformational characteristics that are closely related to the selectivities towards μ/§ opioid receptors. With the use of the proton‐proton distances derived from ROESY measurements in aqueous solution, 50 possible 3D structures are generated by means of distance geometry calculations. The conformers which satisfy the distance constraints and the torsion angles estimated from JNHCxHvicinal coupling constants within the allowable range are then subjected to molecular dynamics simulations for 10 ps after equilibration. Although dermorphin and deltorphin‐II are both in equilibrium among many flexible conformers, some conformational differences are observed between these peptides: many conformers of dermorphin show a structure rounded at theN‐terminal Tyr‐D‐Ala‐Phe‐Gly‐Tyr andC‐terminal Gly‐Tyr‐Pro‐Ser‐NH2moieties, which are almost at right angles to each other, while those of deltorphin‐II are characterized by a ‘hook’ ‐shaped backbone structure in which the nearly extended conformation of the Val‐Val‐Gly‐NH2sequence is located under the folded conformation of theN‐terminal Tyr‐D‐Ala‐Phe‐Glu sequence. The possible relationship between these conformational characteristics and the μ/§‐opioid receptor selectivities is discussed.
A new synthesis of Rink's polymer, 4‐(2′,4′‐dimethoxyphenylhydroxymethyl) phenoxymethylpolystyrene
Authors: VÁRADI GYÖRGYI , TÓTH GÁBOR K. , PENKE BOTOND
Published: August 2010

For solid‐phase peptide synthesis, 2, 4‐dimethoxy‐4′ ‐hydroxybenzhydrol linker was prepared via lithium borohydride reduction of 2, 4‐dimethoxy‐4′‐hydroxybenzophenone. The potassium salt of the linker was coupled to chloromethylpolystyrene. This method proved to be better than use of the cesium salt. This new synthesis gave a polymer with appropriate structure and a good degree of substitution.
Direct assignment of disulfide bonds by Edman degradation of selected peptide fragments
Authors: HANIU MOITSURU , ACKLIN CHRISTINE , KENNEY WILLIAM C. , ROHDE MICHAEL F.
Published: August 2010

Disulfide linkages in peptides or proteins were analyzed by automated gas‐phase Edman sequencing performed in minimized reducing agents. If cystine linkage was regulated at the same position in two peptides during peptide preparation, the diphenylthiohydantoin derivative of cystine was significantly recovered by Edman reaction. In contrast, when the crosslinked half cystines were present at different positions in the peptides, the derivative could be poorly detected. Upon direct sequence analysis of intact bovine insulin, the PTH derivatives of cystine from both Cys‐A7 and Cys‐B7 were significantly released after Edman cycle 7 and gave approximately 20% recovery of common PTH amino acids. However, Cys‐Al1 linked to Cys‐A6 was poorly detectable after Edman cycle 11. For general use of this method, proteins need to be subjected to several sets of proteolytic or chemical cleavages in the hope that at least one of the fragments will have cystine linkage at the same position. This method was applied to several fragments of platelet‐derived growth factor B chain and brain‐derived neurotrophic factor.
Relevance of sequence statistics for the properties of extremophilic proteins
Authors: BÖHM GERALD , JAENICKE RAINER
Published: August 2010

The amino acid composition of proteins from mesophilic and extremophilic organisms is commonly assumed to reflect the mechanisms of molecular adaptation to extremes of physical conditions. In this context, halo‐philic behaviour has been attributed to significantly increased numbers of aspartic and glutamic acid residues. However, extending the analysis to a statistically relevant set of related proteins, dihydrofolate reductase from Halobacterium volcanii, as an example, shows that the increase in negative charge is found to be less significant than other exchanges of amino acids (e.g., Ala, Am, Arg, Lys, Phe, Ser). Thus, the high water binding capacity of negatively charged residues cannot be unambiguously correlated with the anomalous stability of halophilic proteins.

A similar caveat holds for generalizations regarding the thermal stability of proteins. In this case, D‐glyceraldehyde‐3‐phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima was compared with a number of mesophilic and moderately thermophilic homologs. Again, ‘traffic rules of stabilization’, in terms of amino acid changes in going from mesophilic to thermophilic proteins, cannot be given.
Interaction of calmodulin with synthetic deletion peptides of melittin
Authors: LIU LIPING , YAN HUSHENG , NI AIGUO , CHENG XIAOHUI , HE BINGLIN
Published: August 2010

The 26‐residue peptide melittin present in bee venom has been shown to bind calmodulin tightly. In this study we synthesized the following series of deletion peptides of melittin by the solid‐phase method: Mel12, Mel13, Mel 14, Mel 15, Mel15F. The results of this study show that the deletion peptides Mel 14 and Mel 15 have almost the same binding activity as the intact native peptide. Each deletion peptide forms a 1:1 complex with calmodulin according to electrophoresis analysis. When the tryptophanyl residue of Mel15 was replaced by the phenylalaninyl residue, the dissociation constant of the peptide—calmodulin complex increased. This shows the importance of the tryptophanyl residue for binding to calmodulin.
Kinetics of acid‐catalyzed degradation of cyclosporin A and its analogs in aqueous solution
Authors: OLIYAI REZA , SAFADI MUHAMMAD , MEIER PATRICK G. , HU MING‐KUNG , RICH DANIEL H. , STELLA VALENTINO J.
Published: August 2010

The kinetics and mechanism of the degradation of cyclosporin A have been studied under aqueous acidic conditions. The rate of degradation was found to be specific acid‐catalyzed over the pH range studied (1–4), with isocyclosporin A as the predominant degradation product. Selective reduction of the olefinic bond of the amino acid 2‐N‐methyl‐(R)‐((E)‐2‐butenyl)‐4‐methyl‐l‐threonine (MeBmt) did not affect the overall degradation kinetics and product distribution of cyclosporin A. These observations indicate that the alternative degradation pathway involving intramolecular alkoxy addition to the olefinic bond of amino acid MeBmt apparently does not significantly contribute to the overall degradation kinetics of cyclosporin A in the pH range 1–4. The chemical reactivity of O‐acetyl‐cyclosporin A was examined to probe the governing mechanism for the isomerization of cyclosporin A. Under identical conditions, O‐acetyl‐cyclosporin A showed a much greater chemical stability than cyclosporin A, consistent with a mechanism involving the hydroxyoxazolidine intermediate. The chemical stability of cyclosporin C, which contains two β‐hydroxyl groups, was also examined. The rate and product distribution for the degradation of cyclosporin C suggest that under aqueous acidic conditions it undergoes N,O‐acyl migration solely at the amino acid residue MeBmt. Additionally, the impact of side‐chain bulkiness of amino acid MeBmt was examined by studying the degradation kinetics of a series of cyclosporin A analogs. The apparent rate constants for the isomerization of these analogs were not significantly different from that of cyclosporin A, indicating that the terminal bulkiness of amino acid MeBmt may not play a critical role in controlling the rate and extent of N,O‐acyl migration in cyclosporin A.
The concept of superactive esters
Authors: KAMIŃSKI ZBIGNIEW J.
Published: August 2010

According to the concept presented, esters forming an amide (peptide) bond by the mechanism S#nDN or S#*NDN involving fast decay of the tetrahedral intermediate may behave as ‘superactive acylating reagents’. These should render coupling involving less reactive substrates, i.e. sterically hindered or ‘difficult coupling sequences’, much faster and more uniform than classic active esters. In extremely cases this advantage could be significant, and the calculated increase in time required for 99.9% coupling substrates 6 pKa units less reactive than the standard ones reaches 2512000 τ1/2 units for classic active esters, but only 631 τ1/2 units for reaction involving ‘superactive esters’. The postulated change of mechanism is expected for esters bearing a leaving group which is able to undergo an additional, synchronous, energetically favored process accompanying its departure, as has been observed in the case of triazine esters. Some advantages of triazine superactive esters in the condensation of sterically hindered substrates are demonstrated.
Modification of arginine residues in ovine prolactin by 1,2‐cyclohexanedione
Authors: CYMES GISELA D. , ATLASOVICH FABIÁN M. , CARIDAD JUAN J. , IGLESIAS M. MERCEDES , WOLFENSTEIN‐TODEL CARLOTA
Published: August 2010

The reactivity of arginine residues in ovine prolactin was studied by reaction with 1,2‐cyclohexanedione. Kinetic analysis of the data showed a good fit with two simultaneous pseudo‐first‐order equations with apparent velocity constants of 0.28 and 1.2 × 10−2 min−1, corresponding to 1.8 ‘fast’ and 8.7 ‘slow’ residues, respectively. Modification led to a decrease in binding capacity to lactogenic rat liver receptors, and apparently the modification of the two ‘fast’ reacting arginine residues is responsible for the rapid loss of this capacity.

The presence of a non‐reacting arginine has been described in human and bovine growth hormones, and it is located near the carboxy‐terminus. This lack of reactivity is probably due to the formation of a salt bridge, since the arginine residue becomes susceptible to modification once the peptide is separated from the rest of the molecule. This salt bridge is absent in ovine prolactin, since the homologous arginine residue is reactive with cyclohexanedione. This result suggests that there could be a difference between the three‐dimensional structure of ovine prolactin and of the growth hormones, at least near the carboxy‐terminal region of the molecule.
Activation of carboxylic acids by pyrocarbonates. Synthesis of arylamides of N ‐protected amino acids and small peptides using dialkyl pyrocarbonates as condensing reagents
Authors: POZDNEV V.F.
Published: August 2010

Activation of carboxylic acids was achieved via dialkyl pyrocarbonates (ROCO)2O, (R = ethyl, isopropyl, sec‐butyl, tert‐butyl) in aprotic solvents in the presence of tertiary amines. A convenient one‐pot procedure for the preparation of arylamides from N‐protected amino acids including arginine and from di‐tert‐butyl pyrocarbonate in the presence of pyridine (Boc2O‐pyridine system) was reported. Analogously, diisopropyl, di‐sec‐butyl or diethyl pyrocarbonate could be used in the presence of N‐methylmorpholine or triethylamine. A wide variety of N‐protected amino acid arylamides were prepared in good yields.
Problematic sequences in the synthesis of G‐protein peptides
Authors: STEINSCHNEIDER ASSAF
Published: August 2010

Peptide synthesis is hampered by amino acid sequence‐dependent aminoacylation (coupling) difficulties that are only partially understood. Analysis of coupling efficiencies in Fmoc‐based, solid‐phase synthesis of G‐protein fragments revealed that several problematic regions included a tetrapeptide structural motif consisting of (in the order of synthesis): (1) an aliphatic amino acid residue, (2) Asp, (3) and (4) a polar, H‐bonding residue each. The results suggest that interference with aminoacylation involved residue‐specific interactions, probably akin to those acting in protein‐protein adhesion, that occurred between functional groups at the reaction center and others located elsewhere in the peptide molecule. Difficult couplings did not correlate in any meaningful way with conformationally based predictive parameters in the literature. The present investigation points towards the occurrence of putative adhesion signals in intact G‐protein α‐subunits where their sequences are highly conserved, suggesting biological function.
Delta opioid receptor selective ligands; DPLPE‐deltorphin chimeric peptide analogues†
Authors: MISICKA ALEKSANDRA , LIPKOWSKI ANDRZEJ W. , HORVATH ROBERT , DAVIS PEG , PORRECA FRANK , YAMAMURA HENRY I. , HRUBY VICTOR J.
Published: August 2010

Further efforts to correlate the topography of the bioactive structures of DPDPE and the deltorphins, two δ‐opioid receptor active peptide families, are reported. A number of DPLPE‐deltorphin chimeric peptides have been synthesized in which the C‐terminal dipeptide δ‐address of the deltorphins (‐Val‐GlyNH2, ‐Nle‐GlyNH2) have been linked to the highly δ‐opioid selective cyclic peptides DPDPE or DPLPE. These studies demonstrate that a major structural feature determining high potency of hybrid analogues is the chirality of the amino acid residue in position 5. The radioligand binding assays have revealed a decrease in potency (compared to DPDPE) at §‐receptors when the C‐terminal dipeptides were added to DPDPE. On the other hand, chimeric peptides of DPLPE with these same C‐terminal dipeptides retained high δ‐selectivity and affinity. Similar results were obtained using the mouse vas deferens (MVD) and guinea pig ileum (GPI) bioassays. The importance of the hydrophilicity of amino acids in positions 2 and 5 for δ‐selectivity is consistent with the previous finding for DPLPE and DPDPE. On the other hand, the replacement of phenylalanine‐4 with p‐chlorophenylalanine‐4 did not increase δ‐selectivity as in DPDPE. These findings suggest that the δ‐receptor interacts with hybridized enkephalins and deltorphins somewhat differently than with DPDPE.
Different sequence environments of amino acid residues involved and not involved in long‐range interactions in proteins
Authors: TÜDÖS ÉVA , FISER ANDRÁS , SIMON ISTVAN
Published: August 2010

No method has yet been available to decode information, hidden in the protein primary structure, on long‐range interactions of amino acids. Even a limited amount of information on long‐range interactions could help in conformational energy calculations of protein structures and could lead to a better understanding of how the primary structure of proteins determines their conformation.

The sequence environments of amino‐acid residues were compared from the viewpoint of their participation in long‐range interactions. By using the simplest definition, residues were considered as partners in a long‐range interaction if they were at least 20 residues apart in the sequence and their Cα, distance was less than 7 Å.

In spite of this rather crude definition, an analysis of 88 unrelated proteins has shown that the sequence environments (10 residues on each side) of those amino acids which are involved in long‐range interactions and of those which are not are significantly different according to the criteria of mathematical statistics. Moreover, in many cases the differences are so pronounced that the involvement of a given amino acid in long‐range interactions can be predicted from its sequence environment.
Conserved and novel structural characteristics of enantiomorphic Leu‐enkephalin
Authors: DOI MITSUNOBU , ISHIBE ATSUO , SHINOZAKI HUMIYOSHI , URATA HIDEHITO , INOUE MASATOSHI , ISHIDA TOSHIMASA
Published: August 2010

The crystal of the Leu‐enkephalin raceinate (L‐Tyr‐Gly‐Gly‐L‐Phe‐L‐Leu and d‐Tyr‐Gly‐Gly‐D‐Phe‐D‐Leu) was obtained as a centrosymmetric space group. Crystal data: C28H37N5O7· 1.5H2O, Mw, = 582.6, triclinic, space group PI, a = 11.176(3), b = 16.115(3), c = 10.204(4) Å, α= 92.41(3), β= 104.86(2), γ= 85.35(2) °, V= 1770(1) Å3, Z= 2; F(000) = 640, μ(CuKα) = 6.50 cm−1, Dx= 1.081 g cm−3. The structure was determined by X‐ray diffraction. The conformation of the Leu‐enkephalin racemate was classified into the extended form which has been often observed in natural enkephalin. The symmetry‐related molecules were connected by hydrogen bonds and arranged in an antiparallel fashion. The molecular packing showed a sheet structure similar to that of natural enkephalin.
AP‐811, a novel ANP‐C receptor selective agonist
Authors: KOYAMA SATOSHI , INOUE TAKESHI , TARAL TAKAO , TAKIMOTO KOICHI , KATO MASAYUKI , ITO KIYOTAKA , NEYA MASHAHIRO , SEKI JIRO , KOBAYASHI YUJI , KYOGOKU YOSHIMASA , YOSHIDA KEIZO
Published: August 2010

AP‐811 is a derivative of the Phe8‐Ile15 region of atrial natriuretic peptide (ANP) and is one of the smallest linear ligands for ANP receptors. The binding and agonist activities of AP‐811 have been compared with those of other ANP analogs for the ANP‐A and ANP‐C receptors. AP–811 binds with a high binding affinity to and is a strong agonist for the ANP‐C receptor, indicating that the binding and agonist sites for this receptor are the same or near each other in the ANP sequence. In contrast, AP‐811 showed no agonistic effect for the ANP‐A receptor, although it could bind to this receptor. Comparing the biological activities of AP‐811 with those of other ANP analogs, we propose that the binding and agonist sites for the ANP‐A receptor may consist of separate regions of ANP. In conclusion, AP‐811 is the smallest C‐receptor‐selective agonist.
A novel strategy for the synthesis of the cysteine‐rich protective antigen of the malaria merozoite surface protein (MSP‐1)
Authors: SPETZLER JANE C. , RAO CHANG , TAM JAMES P.
Published: August 2010

The most promising antigen for a protective malaria vaccine is a cysteine‐rich domain at the carboxyl terminus of the merozoite surface protein (MSP‐1). Passive transfer of anti‐MSP‐1 antibody or immunization of MSP‐1 against infection challenge confers protection in primate and rodent models. The antigen belongs to the three‐disulfide epidermal growth factor (EGF) family based on the alignment of the six cysteines. In the K1 strain there are, however, only four cysteines corresponding to the four carboxyl cysteines of EGF. Furthermore, disulfide pairing would produce a non‐EGF pattern. Because this cysteine‐rich antigen is conformation‐dependent, and reduction of the disulfide bonds abolishes antigenicity, we used a synthetic analog to investigate the probable disulfide pairing of this antigen. This paper describes the synthesis, folding and disulfide pairings of two 50‐residue cysteine‐rich peptides. One contains two disulfides (VK‐50) derived from the native sequence of MSP‐1 of the Thailand K1 strain (aa 1629–1679). The other contains an EGF‐like, three‐disulfide [Cys‐9,14]VK‐50 peptide. Both peptides were synthesized by a solid‐phase method using Fmoc‐chemistry. The crude peptide of VK‐50 was folded, and the disulfide was oxidized by the DMSO method to obtain a structure with an expected disulfide pairing of 3–4, and 5–6. The specific pairing pattern of 1–3, 2–4 and 5–6 in [Cys 9,14]VK‐50 corresponding to EGF in [Cys 9,14]VK‐50 was obtained using a ‘knowledge‐based’ (KB) strategy for their formation. Purified VK‐50 and [Cys‐9,14]VK‐50 had the correct molecular weight, as shown by Cf‐252 fission ionization mass‐spectrometry. The disulfide pairings were confirmed by enzymatic digestion. These two peptides can be conjugated to the multiple peptide antigen core for immunization. The immunological results will allow us to conclude the correct disulfide pairing and the conformational importance of this antigen.
Some ‘difficult sequences’ made easy
Authors: HYDE CAROLYN , JOHNSON T. , OWEN D. , QUIBELL M. , SHEPPARD R.C.
Published: August 2010

Case studies of the solid‐phase synthesis of some ‘difficult sequences’ using continuous‐flow Fmoc‐polyamide techniques are presented. Solvent change and peptide side chain and backbone modification procedures recently found to reduce association effects are applied, and the results compared to syntheses under standard conditions.
Substrate phosphorylation capacities of the major tyrosine protein kinase from the human promyelocytic cell line, HL‐60
Authors: ERNOULD ANNE‐PASCALE , FERRY GILLES , BARRET JEAN‐MARC , GENTON ANNIE , BOUTIN JEAN A.
Published: August 2010

The major tyrosine protein kinase, HPK40, isolated from HL‐60, the preparation of which is described elsewhere (Ernould, A.P., Ferry, G., Barret, J.M., Genton, A. and Boutin, J.A., Eur. J. Biochem., 214, 503–514), was investigated as to its specificity on a number of peptides and proteins. It was found that HPK40 can phosphorylate histones (except histone H4), casein, acid‐treated enolase, actin and tubulin but not calmodulin. Phosphorylation specificity of HPK40 was investigated using over a hundred peptidic structures. HPK40 is not related to the ‘src’ family and does not phosphorylate efficiently either the tetrapeptide NEYT derived from the pp60src autophosphorylation domain or the corresponding peptide RRsrc, RRLIEDNEYTARG. VALYDYESR from the SH3 domain of pp60c‐src is recognized as a substrate with a high phosphorylation level. DEDYIQD, derived from the phosvitin/casein kinase II, was also highly phosphorylated. In order to determine the minimal recognition sequence of HPK40, the phosphorylation of about 60 dito tetrapeptides was investigated. Some of the tetrapeptides, such as *EEYE and NEYE, were well phosphorylated. Even some tripeptides, such as EYE, DYM, TYS and KYE, were recognized by HPK40, while none of the tested dipeptides was recognized as substrate. Sequences of peptides from DRVYHPF (angiotensin), LEEEEEAYGWMDF (minigastrin) and QEEYSAM (from H‐rasl) were examined as substrates. The presence of one or several acidic residues on the Nα‐side of tyrosine residue was identified as the only apparently favorable determinant. These results are steps towards the minimum recognition sequence, which in turn will serve as a lead for chemical modifications in view of obtaining a specific, low‐molecular‐weight, inhibitor of this human tyrosine protein kinase.
Side‐reactions in peptide synthesis
Authors: FORINO ROMUALDO , GALANTINO MAURO , CIPRANDI FRANCO , BAUMER LUCA , ARLANDINI EMANUELE , CASTIGLIONE ROBERTO DE
Published: August 2010

The impurities which formed in the large‐scale synthesis of THF‐γ2, an immunomodulatory peptide of formula H‐Leu‐Glu‐Asp‐Gly‐Pro‐Lys‐Phe‐Leu‐OH, were identified by a combination of analytical methods, and their structure confirmed by synthesis. Most impurities originated from side‐reactions involving the aspartyl residue (cyclization, β‐aspartyl formation and cleavage). Based on this knowledge, modifications were introduced into the work up and the purification procedure which resulted in a very pure final product (>99% by RP‐HPLC).
Study of tensioactive properties of casein signal peptides and their interactions with phospholipids
Authors: CREUZENET C. , HAERTLÉ T.
Published: August 2010

The high degree of sequence conservation in casein signal peptides reflects their unique functional properties. A series of casein signal peptides and derivatives was synthesized in order to study their insertion in phospholipidic mono‐ and bilayer structures. Most of these amphiphilic peptides were found to be highly tensio active. Their conformations differ and are solvent dependent. Fluorescence anisotropy measurements showed that all the peptides of the series could interact with dimyristoylphosphatidyl ‐choline and ‐glycerol when mixed with the lipids prior to hydration of the liposomes. The most soluble peptide, P6, was selected for insertion experiments in multilamellar vesicles. Its interaction with liposomes is efficient and rapid, being temperature dependent. On the one hand, the physico‐chemical measures of interactions of signal peptides of casein β and αs2 confirm their mutual genetic relationship, and on the other hand they show the divergence of casein β and αs2 from casein K signal peptide.
BPTI backbone variants and implications for inhibitory activity
Authors: GROEGER CHRISTIAN , WENZEL HERBERT R. , TSCHESCHE HARALD
Published: August 2010

Structural variants of BPTI Were synthesized en route an enzymatic‐chemical semisynthesis. The P′1‐ P′2 amide bond of the inhibitor molecule, which, as donor, contributes a hydrogen bond towards trypsin in the enzyme‐inhibitor complex, was replaced by either a ketoniethylene function or an ester bond yielding molecules with inhibitory activity. The two backbone‐mutated BPTI derivatives showed increased dissociation constants of their respective trypsin complexes, obviously due to the lack of a single hydrogen‐bond interaction in the enzyme‐inhibitor complex.
Characteristic molecular packing in the crystal structure of tert‐butoxycarbonyl‐L‐phenylalanyl‐L‐methionine methyl ester
Authors: DOI MITSUNOBU , IN YASUKO , INOUE MASATOSHI , ISHIDA TOSHIMASA
Published: August 2010

The molecular conformation and association of the peptide Boc‐L‐Phe‐L‐Met‐OMe have been studied in the solid state by X‐ray diffraction. The peptide crystallizes in the orthorhombic system, space group P212121, with cell parameters of a= 9.821(2), b= 25.394(6), c= 28.714(8) Å3, V= 7161(3) Å3. The structure has been solved by direct methods and refined to a final R of 0.079 for 5464 independent reflections with Fo≥σ(Fo). The crystal consists of three independent molecular conformations per asymmetric unit. Respective peptide backbones adopt an extended conformation with the side‐chains of Phe and Met residues being arranged below and above the backbone chains. Contrary to the sheet structure most frequently observed in the crystal packing of the extended peptide conformations, three independent molecules lie spirally along the c‐axis and form a pin‐wheel‐like crystal packing. The sheet structures formed by two of three independent molecules are almost at right angles to the backbone of the remaining molecule. This molecular packing mode would provide a possible interaction model between the intersecting β‐sheet structure and single‐strand structure of polypeptide. © Munksgaard 1994.
Peptic proteolysis of esterified β‐casein and β‐lactoglobulin
Authors: BRIAND LOÏC , CHOBERT JEAN‐MARC , HAERTLÉ TOMASZ
Published: August 2010

Moderate esterification induces slight secondary structure changes in two major milk proteins, β‐lactoglobulin and β‐casein. Esterification of β‐lactoglobulin prompts its tertiary structure‘melting′, opening it to peptic cleavage. Twenty‐two new cleavage sites were characterised in β‐lactoglobulin and five in β‐casein. Some of them are due to esterification‐improved peptide bond accessibility, some to the bias of pepsin specificity by glutamate and aspartate esters. The resulting fragmentation yields original and partially amphiphilic peptide populations.
Synthesis of a mixture of cyclic peptides based on the Bowman‐Birk reactive site loop to screen for serine protease inhibitors
Authors: DOMINGO GONZALO J. , LEATHERBARROW ROBIN J. , FREEMAN NEIL , PATEL SHILA , WEIR MALCOLM
Published: August 2010

A peptide mixture containing 21 peptide sequences has been constructed to test the Bowman‐Birk inhibitor reactive‐site loop motif as the basis of inhibition for a range of serine proteases. The 21 peptides are all based on an 11 amino acid sequence designed from a Bowman‐Birk like inhibitor reactive‐site loop. Variation has been introduced at the P1 site of the loop, which has been randomised to include all the natural l‐amino acids (except for cysteine), plus the non‐natural l‐amino acids ornithine and norleucine. The mixture of peptides was screened for specific binding to immobilised porcine pancreatic elastase, subtilisin BPN′, α‐chymotrypsin, trypsin, anhydro‐α‐chymotrypsin and anhydrotrypsin. Five peptides from the mixture bind to α‐chymotrypsin, two of which also bind to anhydro‐α‐chymotrypsin, and two peptides bind trypsin, neither of which binds to anhydro‐trypsin. The competitive inhibition constants (K1) and the rates of proteolytic hydrolysis of the individual peptides with their respective enzymes were determined. The rates of hydrolysis were found to vary widely and show little correlation with the K1 values. In the case of the α‐chymotrypsin inhibitors, the peptides with the lowest Ki (0.1–0.05 mM) were the only peptides that bound to anhydro‐α‐chymotrypsin. However, no peptides bound to anhydrotrypsin, suggesting a fundamental difference in the way that α‐chymotrypsin and trypsin are inhibited by these cyclic peptides.
Isolation and characterization of melanotropins from lamprey pituitary glands
Authors: TAKAHASHI AKIYOSHI , AMEMIYA YUTAKA , NOZAKI MASUMI , SOWER STACIA A. , JOSS JEAN , GORBMAN AUBREY , KAWAUCHI HIROSHI
Published: August 2010

Three peptides containing the melanotropin‐core amino‐acid sequence, YXMXHFRWG, were isolated from the pituitary glands of a modern representative of the most primitive vertebrates, the sea lamprey, Petromyzon marinus. MSH‐A, a nonadecapeptide (NPELYQMNHFRWGQPPTHF), is free at both ends. MSH‐B, an eicosapeptide (VQES ADG YRMQHFRWGQPLP), is free at the N‐terminus and amidated at the C‐terminus. They differ strikingly from gnathostome MSHs in structure. The third peptide, with an apparent molecular weight of 15 kDa, was tentatively designated lamprey ACTH, based on a structural feature; the N‐terminal 22‐residue‐MSH (SVSSPKYAMGHFRWGSPDKATI) is followed by four consecutive basic amino acids (RKRR) and a ACTH‐like sequence (PVRPNTSDSPEIPDYAF–). MSH‐B is 10 and 100 times more potent than α‐MSH and MSH‐A, respectively, in a frog skin assay in vitro, whereas the lamprey ACTH showed no melanotropic activity. Lamprey ACTH did, however, show corticotropic activity on the lamprey pronephric and mesonephric tissue.