bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of secondary structure on the rate of deamidation of several growth hormone releasing factor analogs
Authors: STEVENSON CYNTHIA L. , FRIEDMAN ALAN R. , KUBIAK TERESA M. , DONLAN MARY E. , BORCHARDT RONALD T.
Published: August 2010

The objective of this study was to determine whether the rates of deamidation of Asn8in selected growth hormone releasing factor (GRF) analogs were related to the peptide's secondary structures in solution. Bovine or human [Leu27]GRF(1–32)NH2(both having Gly at position 15), [Ala15Leu27]bGRF(1–32)NH2and [Pro15Leu27]bGRF(1–32)NH2were used as model peptides. The peptide helical content (assessed by CD) increased with the increasing methanol concentration and was as follows: 7, 12 and 18% in 0% MeOH; 24, 48 and 52% in 40% MeOH; and 41, 77 and 81% in 80% MeOH for Pro15Leu27bGRF(1–32)NH2, [Leu27]hGRF(1–32)NH2, and Ala15Leu27bGRF(1–32)NH2, respectively. 2D NMR studies done in the presence of 40% CD3OH indicated more helical structure for the Ala15analog as compared to [Len27]hGRF(1–32)NH2. In both these peptides Asn8was included in the helical region. In contrast, the lack of conformational information for the Pro15analog indicated little helical structure around Asn8. The peptides’ deamidation rates decreased and their half‐lives increased with increasing MeOH concentrations. At 40% MeOH, the least helical Pro15bGRF analog (t1/2= 10.78 h) deamidated 1.5 and 2 times faster than its Gly15(t1/2= 15.74 h) and Ala15(t1/2= 21.53 h) counterparts, respectively. This study indicates that helical environment around Asn8in GRF makes this residue less prone to deamidation.
Studies on the disproportionation of mixed anhydrides of N‐alkoxycarbonylamino acids
Authors: BENOITON N. LEO , LEE YOUNG C. , CHEN FRANCIS M.F.
Published: August 2010

Mixed anhydrides from Z‐valine and ethyl, isobutyl, isopropyl and isopropenyl chloroformate, and from N‐tert‐butoxycarbonyl‐ (Boc) and N‐benzyloxycarbonyl‐ (Z) leucine and phenylalanine and Boc‐ and N‐9‐fluorenylmethoxycarbonyl‐valine and ethyl chloroformate were purified and left in dichloromethane at 23°C. The symmetrical anhydride (SyAn) generated after 24 h was determined by normal phase high‐performance chromatography. No SyAn was produced from the anhyrides of Z‐ and Boc‐valine; SyAn was produced from the other anhydrides. Anhydrides examined without isolation generally produced more SyAn than the pure compounds. More SyAn was generated in dimethylformamide and tetrahydrofuran than in dichloromethane, or in the presence of an excess of N‐methylmorpholine or triethylamine. The anhydride from isobutyl chloroformate was much more stable than the anhydride from ethyl chloroformate. It is suggested that disproportionation of pure mixed carboxylic acid – carbonic acid anhydrides occurs by a bimolecular mechanism.
Temporary serine protection in solid phase synthesis of LH‐RH analogs
Authors: ARZENO H.B. , BINGENHEIMER W. , BLANCHETTE R. , MORGANS D.J. , ROBINSON J.
Published: August 2010

Methodology for the large‐scale, solid‐phase synthesis of nafarelin, I, an LH‐RH agonist and RS‐26306, III, and LH‐RH antagonist, is described. Nα‐Boc protected amino acids were used in the synthesis. The only side‐chain‐protected amino acids required were BocHis(Tos)‐OH and BocSer(t‐Bu)‐OH. The use of temporary protection on serine eliminates the formation of bis‐serine derivatives (II and IV), which presents a major limitation to the use of minimal protection schemes for large‐scale synthesis. Using this approach, the side‐chain protecting groups are cleaved during the synthesis, and HF deprotection in a separate step is not required.
Chemical synthesis of a designed β‐protein through the flow‐polyamide method
Authors: BIANCHI E. , SOLLAZZO M. , TRAMONTANO A. , PESSI A.
Published: August 2010

A designed, 61‐residue long, metal‐binding protein was synthesized through the flow‐polyamide method. This protein, named Minibody (Mini‐antibody), contains a β‐sheet scaffold and two regions corresponding to immunoglobulin hypervariable loops (H1 and H2), onto which a metal binding site was engineered. The protein is extremely hydrophobic, with a 70%β structure, Accordingly, it was anticipated, and actually found, to be a “difficult sequence” for all its length. Comparison of the standard, “minimum redundancy” protocol (single coupling, low excess of activated species) with a different protocol (double and triple coupling plus capping) showed that the two produced approximately the same amount of full‐length product (3.7% after extensive purification). Capping was effective in blocking the unreacted amino groups, converting most failure sequences into truncated ones, a possible aid to implement affinity‐type chromatographic protocols. Purification was complicated by the very low solubility of the molecule, coupled to a high tendency to aggregate even in concentrated chaotropic media (8 M urea). Nevertheless, a multi‐dimensional purification scheme produced a highly homogeneous product, with the expected IonSpray mass spectrum. The Minibody produced by recDNA methods showed identical chromatographic, spectroscopic and biochemical properties to those of the synthetic product.
Synthesis of potent antagonists of substance P by modifying the methionyl and glutaminyl residues of its C‐terminal hexapeptide and without using d‐amino acids
Authors: MANOLOPOULOU AMALIA , KARAGIANNIS KOSTAS , STAVROPOULOS GEORGE , POULOS CONSTANTINE , JORDAN CHRISTOPHER C. , HAGAN RUSSELL M.
Published: August 2010

Analogues of [Orn6]‐SP6–11 have been synthesized in which the Met11‐NH2 residue is replaced by the α,γ‐dimethyl, α,γ‐dibenzyl and α,γ‐di‐tert‐butyl esters of glutamic acid. These analogues were tested in three in vitro preparations representative of NK‐1, NK‐2 and NK‐3 receptor types for agonist and antagonist activity. The dimethyl analogue is a selective full agonist in the NK‐1 receptor type and a weak antagonist in the other two receptor types, while the dibenzyl and the di‐tert‐butyl analogues are potent antagonists in the NK‐1 receptor type and weak antagonists in the other two receptor types. It is concluded that appropriate modification at the α‐carboxamide and the side chain of the methionine residue of substance P may induce antagonism without using d‐amino acids.
Conformation of dehydropeptides
Authors: GUPTA ALKA , CHAUHAN V.S.
Published: August 2010

Six model dipeptide methyl amides containing dehydroaminobutyric acid (Δ Abu) of the type Boc‐X‐δZAbu‐NHCH3 and Box‐X‐δEAbu‐NHCH3, X = Ala, Val, Phe (Boc =tert‐butoxycarbonyl), have been synthesized and their solution conformations explored using 300 MHz 1H NMR and IR spectroscopy. Studies based on delineation of intramolecularly hydrogen bonded NH groups in CDCl3 and (CD3)2SO revealed that none of the NH groups is appreciably solvent shielded. Difference NOE (Nuclear Overhauser Effect) studies have also failed to detect the presence of any discernible turn structure in these peptides. These studies indicate that the conformational preferences of peptides containing, α, β‐dehydroaminobutyric acid are different from those of ΔZPhe and ΔZLeu. It appears that steric interactions due to the β‐substituent in the dehydroamino acid moiety play an important role. Unlike ΔZPhe and ΔZLeu, which have relatively large β‐substituents, phenyl and isopropyl, respectively, and stabilize a β‐turn, the β‐methyl group of ΔZAbu or ΔEAbu is readily accommodated in extended conformation. Clearly, the size of β‐substituent in dehydroamino acid crucially influences the conformational preferences. Thus, it may be possible to use different dehydroamino acids to introduce variable but definite constraints in synthetic peptides.
SCF‐MO study of the polyhydration of N,N′ ‐dimethylmalonamide
Authors: ALEMÁN CARLOS , PEREZ JUAN J.
Published: August 2010

The effect of hydration on the conformation of the N,3 nylons has been studied using AM1 SCF‐MO on the model molecule N,N′‐dimethylmalonamide (NNMA). The polyhydration model has been used, so that only the water molecules directly bonded to solute molecule are considered. The study reveals that, although hydration induces dramatic changes in the torsional angles of the molecule, the new conformation still remains in the helical region of the Ramachandran map.
Conformations of cyclic pentapeptide endothelin receptor antagonists
Authors: BEAN JOHN W. , PEISHOFF CATHERINE E. , KOPPLE KENNETH D.
Published: August 2010

The solution conformations in methanol and chloroform of the endothelin A receptor antagonists cyclo(dV‐L‐dW‐dD‐P), 1, and cyclo(dV‐Nα‐MeL‐dW‐dD‐P), 2, have been studied by NMR spectroscopy at room temperature and below. In these solvents, both peptides were found to have a well defined peptide backbone conformation composed of a type II β turn at the Leu‐D‐Trp and a γ′ turn at Pro. This conformation is in agreement with results reported for 1 in other solvents and consistent with the expected location of the N‐methyl substituent in that backbone. In methanol, both peptides show NOE and chemical shift evidence of close contact between the Leu and D‐Trp side chains. This interaction is greatly reduced or absent in chloroform, and is stronger in methanol at 203 K than at 298 K.
Design and synthesis of novel inhibitors of prohormone convertases
Authors: BASAK AJOY , JEAN FRANCOIS , SEIDAH NABIL G. , LAZURE CLAUDE
Published: August 2010

Prohormone convertase‐1 (PC1) and furin are subtilisin‐like endopeptidases involved in the biosynthesis of peptide hormones. Five decapeptides representing the junction between the pro‐region and the catalytic region of PC1 were prepared. The core sequence corresponded to D‐Tyr‐Arg‐Ser‐Lys‐Arg‐Xaa‐Val‐Gln‐Lys‐Asp where D‐Tyr replaces the native Glu residue and Xaa, representing the P1′ position, corresponds to L‐Ser, L‐Leu or the unnatural amino acids, D‐Ser, β‐Ala, γ‐Abu, β‐Cha or γ‐Hyp. Another analog incorporating an Orn residue in place of the Arg at the P1 site was also prepared. These peptides, synthesized by solid‐phase Fmoc chemistry, were fully characterized by FAB‐MS, 1H‐NMR and amino acid composition. Except for Orn, γ‐Hyp, L/D‐Ser and L‐Leu containing analogs, the others were found to be moderate to potent competitive inhibitors of hPCl activity in the following order: γ‐Abu>β‐Cha>β‐Ala, with Ki values ranging from 1 to 8.6 μM. Both L‐Ser and L‐Leu analogs were correctly cleaved at the acyl carbon COOH‐terminal to the Lys‐Arg pair by human PCl, whereas β‐Cha, γ‐Abu, β‐Ala and D‐Ser analogs proved to be very poor substrates. The Orn and γ‐Hyp derivatives were not cleaved by the enzyme at all. The three analogs containing β‐Cha, γ‐Abu and β‐Ala also proved to be potent inhibitors of the human furin activity in the following order: β‐Ala>β‐Cha> γ‐Abu, with K1 ranging from 0.8 to 2.2μM. Two more peptides, modified at the P1 position by addition of a semicarbazone (SC) moiety, were also synthesized by liquid‐phase chemistry. These peptides, Arg‐Ser‐Lys‐argininal‐SC and Arg‐Lys‐Lys‐argininal‐SC, also proved to be potent competitive inhibitors of hPCl and exhibited Ki values 3.6 and 2.3 μM, respectively.
Chemical synthesis and biological activity of the EGF‐like domain of heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF)
Authors: SHIN SONG YUB , TAKENOUCHI TAKATO , YOKOYAMA TETSUO , OHTAKI TETSUYA , MUNEKATA EISUKE
Published: August 2010

Heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF) is a recently discovered member of the epidermal growth factor (EGF) family. This novel growth factor possesses the EGF‐like domain in the carboxyl portion. In order to evaluate the biological function of the EGF‐like domain in HB‐EGF, human HB‐EGF(44–86) corresponding to the EGF‐like domain was synthesized by the solid‐phase procedure using the Fmoc strategy. It was confirmed by amino acid microsequencing of cystine‐containing fragments derived from thermolytic digestion that the pattern of three disulfide bond pairings in synthetic HB‐EGF(44–86) was consistent with that of EGF and transforming growth factor‐α (TGF‐α). The homogeneity of the synthetic peptide was confirmed by analytical RP‐HPLC, amino acid analysis and fast atom bombardment mass spectrometer (FAB‐MS). Compared with h‐EGF, the EGF‐like domain of human HB‐EGF showed a comparable mitogenic activity in the proliferation of NIH/3T3 fibroblast cells. These results suggest that the EGF‐like domain of human HB‐EGF may play an important role in mitogenic activity.
Synthesis of a cyclic analogue of the C‐loop region of epidermal growth factor, containing 1‐aminocyclopropane‐1‐carboxylic acid
Authors: BAILIE JANICE R. , WALKER BRIAN , NELSON JOHN , MURPHY RICHARD F.
Published: August 2010

We report the synthesis of a cyclic analogue of epidermal growth factor sequence 33–42 with substitution of 1‐aminocyclopropane‐1‐carboxylic acid for glycine at position 39 (N‐acetyl‐Cys‐Val‐Ile‐Gly‐Tyr‐Ser‐ACPCA‐Asp‐Arg‐Cys‐NH2). The analogue was synthesised by solid‐phase methods, using t‐Boc chemistry and acid‐labile side‐chain protecting groups. The use of the 4‐methoxybenzyl protecting group for C‐ and N‐terminal cysteine residues resulted in the spontaneous formation of the desired intramolecular disulfide bond after HF deprotection.
Synthetic immunochemistry of glycohexapeptide analogues characteristic of oncofetal fibronectin
Authors: ZHENG M. , GOBBO M. , BIONDI L. , FILIRA F. , HAKOMORI S. , ROCCHI R.
Published: August 2010

Monoclonal antibody FDC‐6, and its second‐generation antibodies FDB‐1 and FDB‐4, are able to distinguish between fibronectin (FN) from fetal or cancer tissue (onco‐FN) vs. FN from normal adult tissue and plasma (nor‐FN). The epitope structure recognized by the above antibodies is the glycohexapeptide H‐Val‐(GalNAc‐α)Thr‐His‐Pro‐Gly‐Tyr‐OH (P2). In order to define further the specificity of the reactive site, we synthesized various glycopeptides based on the unglycosylated hexapeptide sequence (P1) and compared their reactivities with these antibodies. In continuation of our structure‐activity relationship studies the (Asn3,Ala5)‐glycohexapeptide analogue (P3) was synthesized by a solid‐phase procedure. The [Ala(CN)3,Ala3]‐glycopeptide (P4), owing to dehydration of the asparagine side chain amide during carboxyl activation of Fmoc‐Asn‐OH, was also isolated. Fmoc‐[GalNAc(Ac)3‐α]Thr‐OH was used for incorporating the glycosylated amino acid residue. For the sake of comparison the epitope P2 and the hexapeptide sequence P1 were also synthesized. The final products were characterized by elemental and amino acid analyses, optical rotation, analytical HPLC, proton NMR and fast‐atom bombardment mass spectroscopy. Synthetic analogues were applied to inhibit onco‐FN specific MAbs FDB‐1, FDB‐4 and FDC‐6 binding to immobilized onco‐FN. and their activities were compared with onco‐FN and nor‐FN. P2 exhibited an activity similar to that of an intact molecule of onco‐FN. Deglycosylation (P1) or replacement of amino acid (P3, P4) greatly reduced activity. Data clearly showed that P2 was the minimal essential structure of the epitope in onco‐FN defined by MAbs FDB‐1, FDB‐4 and FDC‐6. They also suggested that single glycosylation made this epitope more exposed on an onco‐FN molecule.
Crystal structure analysis of a β‐turn mimic in hydrazino peptides
Authors: AUBRY ANDRE , MANGEOT JEAN‐PAUL , VIDAL JOELLE , COLLET ANDRE , ZERKOUT SAID , MARRAUD MICHEL
Published: August 2010

The crystal structures of four hydrazino peptides (Piv‐Pro‐h(Nα‐Bzl)Gly‐NHiPr 1, Piv‐Pro‐hAla‐NHiPr 2, Moc‐hPro‐NHiPr 3, and Boc‐hPro‐Gly‐N(OH)Me 4) deriving from the hydrazino analogues of glycine (hGly), l‐alanine (hAla) or l‐proline (hPro) have been solved. They reveal a common folded structure of the α‐hydrazino acid residue characterized by a bifurcated hydrogen bond closing an eight‐membered cycle. This folded structure is topologically similar to the βII′‐turn in peptides, and the CO‐NH‐N hydrazide link can be considered as a good turn‐inducer in peptide analogues.
Hydrolysis pattern of procasomorphin by gut proteases from plant parasite Heliothis zea, determined by sequence analyses performed on the unfractionated digestion mixtures
Authors: PETRILLI PASQUALE , CAPORALE CARLO , CARUSO CARLA
Published: August 2010

The digestion pattern of procasomorphin, a putative precursor of beta;‐casomorphins (β‐casein‐derived opioid peptides), by gut proteases from the plant parasite insect Heliothis zea has been determined to evaluate the possibility of introducing in plants the gene encoding for this peptide in order to confer resistance to damage by the insect. The method we used is based on the possibility of deducing the sequence of the fragments produced by proteases at different times of digestion by means of automatic Edman degradation performed on the fragment mixtures without any purification steps. This approach can be considered of general utility in studying the digestion of peptides by a mixture of proteases.
Synthesis of the cardiac inotropic polypeptide anthopleurin‐A
Authors: PENNINGTON MICHAEL W. , ZADENBERG INNA , BYRNES MICHAEL E. , NORTON RAYMOND S. , KEM WILLIAM R.
Published: August 2010

The sea anemone polypeptide anthopleurin‐A (AP‐A) at nanomolar concentrations enhances myocardial contractility without affecting automaticity. It has a therapeutic index higher than that of the digitalis glyco‐sides, and may serve as a molecular model for designing a new class of inotropic drugs acting on the myocardial Na channel at site 3. AP‐A is a 49 residue peptide crosslinked by three disulfide bonds; its tertiary structure has been determined by NMR. Here we report the solid‐phase synthesis of this polypeptide. Synthetic AP‐A displayed CD and NMR spectra identical with those of the natural toxin; it possessed 94±15% of the inotropic activity of natural AP‐A. Therefore, it is feasible to prepare various type 1 sea anemone toxin analogs by solid‐phase chemical synthesis in order to identify side chains important for peptide folding and interaction with sodium channels.
Structure and protein kinase C stimulating activities of lactam analogues of human parathyroid hormone fragment
Authors: NEUGEBAUER W. , GAGNON L. , WHITFIELD J. , WILLICK G.E.
Published: August 2010

Five analogues of human parathyroid hormone (hPTH‐(20‐34)‐NH2, I; cyclo[Lys26‐Asp30]‐hPTH‐(20‐34)‐NH2, II; cyclo[Glu22‐Lys26]‐hPTH‐(20‐34)‐NH2, III; cyclo[Lys27‐Asp30]‐hPTH‐(20‐34)‐NH2, IV; and [Leu27]‐hPTH‐(20‐34)‐NH2, V) were tested for their ability to promote membrane‐bound protein kinase C (PKC) activity in a rat osteosarcoma cell line (ROS 17/2). Analogues I, II and V stimulated PKC activity in the picomolar range, whereas analogues III and IV did not stimulate this activity at any concentration tested. The circular dichroism spectra in neutral, aqueous buffer showed an increase in α‐helix in analogues II, III and V as compared to I; this increase appeared to be in the region of the cyclic lactam structure. Analogue IV did not adopt a helical structure, even in the presence of 40% trifluoroethanol, a helix‐promoting solvent. The remaining analogues showed a three‐ to four‐fold enhancement of α‐helix in this solvent. Analogues II and III had increased retention times in reversed‐phase chromatography, as compared to I and IV, This is consistent with a stabilization of amphiphilic helix in analogues II and III compared with I and IV, The data suggest that in the region bounded approximately by residues 24–32, an amphiphilic α‐helix is important for correct functional binding to the PTH receptor.
Conformational characteristics of asparaginyl residues in proteins
Authors: SRINIVASAN N. , ANURADHA V.S. , RAMAKRISHNAN C. , SOWDHAMINI R. , BALARAM P.
Published: August 2010

Backbone conformations at 1064 asparaginyl residues in 123 non‐homologous, high‐resolution X‐ray structures of proteins were analysed. Asn adopts conformations in left‐handed α‐helical region and other partially allowed regions in the Ramachandran map more readily than any other non‐glycyl residue. Asn conformational clusters in the (φ,ψ) regions of left‐handed α‐helix, right‐handed 7ALPHA;‐helix and extended (β) strands were investigated in detail for their occurrence in various secondary structures, especially in β‐turn regions. Preferences were observed for Asn conformations in different positions in various β‐turn types, including the first and fourth positions of the turn. Asparaginyl residues with extended conformations are found to occur frequently in irregular regions, although they are expected to occur predominantly in extended strands or in the third position of type II β‐turns. Asn conformations at the N‐cap positions of helices strongly prefer extended conformation than α1, which seems to be characteristic of non‐glycyl residues at that position. In the linkers connecting two extended strands and those connecting an α‐helix and an extended strand, Asn with α1 or αR conformation is more favoured than Asn with the β‐conformation. Analysis of Asn‐Asn doublets and Asn‐X‐Asn triplets permitted identification of conformational families in such sequences. Results of this investigation provide useful hints in modelling Asn‐rich regions in proteins such as malaria parasite coat protein.
Peptide synthesis catalyzed by the Glu/Asp‐specific endopeptidase
Authors: BONGERS JACOB , LIU WEN , LAMBROS THEODORE , BREDDAM KLAUS , CAMPBELL ROBERT M. , FELIX ARTHUR M. , HEIMER EDGAR P.
Published: August 2010

We recently described a two‐step enzymatic semisynthesis of the superpotent analog of human growth hormone releasing factor, [desNH2Tyr1,D‐Ala2,Ala15]‐GRF(1‐29)‐NH2 (4), from the precursor, [Ala15,29]‐GRF(4‐29)‐OH (1). C‐Terminal amidation of 1 to form [Ala15]‐GRF(4‐29)‐NH2 (2) was achieved by carboxypeptidase‐Y‐catalyzed exchange of Ala29‐OH for Arg‐NH2. The target analog 4 was then obtained by acylation of segment 2 with desNH2Tyr‐D‐Ala‐Asp(OH)‐OR (3) (R = CH3CH2 or 4‐NO2C6H4CH2) catalyzed by the V8 protease. In this paper we report on the use of the recently isolated Glu/Asp‐specific endopeptidase (GSE) from Bacillus licheniformis, which is shown to be an efficient catalyst for the segment condensation of 2 and 3. GSE is more stable than the V8 protease under the conditions employed (20% DMF, pH 8.2, 37 °C). The extent of conversion of 2 into 4 is limited by proteolyses at Asp3‐Ala4 and Asp25‐Ile26. However, this proteolysis is virtually eliminated by use of the appropriate ester leaving group, R.

A systematic study of the kinetics of the GSE‐catalyzed segment condensations of 2 and a series of tripeptide esters, desNH2Tyr‐D‐Ala‐Asp(OH)‐OR (3) [R = CH3CH2‐ (3a), CH3‐ (3b), ClCH2CH2‐ (3c), C6H5CH2‐ (3d), 4‐No2C6H4CH2‐ (3e)] revealed that the rate of aminolysis versus proteolysis, and hence the conversion of 2 into 4, increase with increasing specificity (Vmax/Km) of GSE for the tripeptide ester. The specificity varies in the order 3e>3d>3c>3b>3a and appears to depend on an increase in the maximum turnover rate (Vmax) with increasing basicity of R. This work demonstrates the coupling of a small peptide segment containing unnatural amino acids to the N‐terminus of an intermediate‐length polypeptide, without side‐chain protection, by GSE, a potentially less costly and more stable alternative to the V8 protease.
Bony fish neurophysins Identification of MSEL‐ and VLDV‐neurophysins of the pollack (Pollachius virens)
Authors: CHAUVET JACQUELINE , LEE WON‐JAE , CHAUVET MARIE‐THÉRÈSE , ACHER ROGER
Published: August 2010

The two types of neurophysins known in vertebrate species, namely MSEL‐neurophysin (vasopressin‐like hormone‐associated neurophysin) and VLDV‐neurophysin (oxytocin‐like hormone‐associated neurophysin) have been purified from the pollack (Pollachius virens) pituitary through a combination of molecular sieving and high‐pressure liquid chromatography (HPLC). Homogeneity has been checked by gel electrophoresis and rerun in HPLC. The apparent molecular masses measured by SDS‐electrophoresis are near 12 kDa, significantly higher than those found for their mammalian homologues (10 kDa). The two types of neurophysins have been recognized through their N‐terminal amino acid sequences. The primary structure of MSEL‐neurophysin has been partially determined using automated Edman degradation applied on native and reduced‐alkylated protein, as well as peptides derived by trypsin or staphylococcal proteinase hydrolyses. Comparison of pollack MSEL‐neurophysin with ox, goose and frog counterparts reveals that particular positions in the polypeptide chain are subjected to substitutions and that the numbers of substitutions do not seem closely related to the paleontological times of divergence between the different vertebrate classes.
Structure‐activity analysis of the antitumor and hemolytic properties of the amphiphilic α‐helical peptide, C18G
Authors: PECK‐MILLER KAREN A. , BLAKE JAMES , COSAND WESLEY L. , DARVEAU RICHARD P. , FELL H. PERRY
Published: August 2010

The in vitro antitumor and hemolytic activities of analogs of peptide C18G were compared in order to elucidate important structural features which affect cytotoxicity. The sequence of C18G, a basic peptide which can form an amphiphilic α‐helix, is a derivative of the carboxyl terminus of human platelet factor IV. The results demonstrate that both amphiphilicity and helicity are essential for peptide activity, and that addition of a negatively charged amino acid results in decreased cell lysis. Whereas peptides exhibiting various degrees of potency did not differ with respect to helical content, an increase in peptide hydrophobicity did correlate with an increase in antitumor and hemolytic activity, as well as susceptibility to inhibition by serum. Higher hydrophobicity could be associated with improved ability to insert into the cell membrane. The position or context of specific residues within an amphiphilic peptide can also be important for activity. Furthermore, an increase in tumoricidal activity is not always accompanied by an increase in hemolytic activity or susceptibility to inhibition by serum. Possible reasons for the lower sensitivity of RBCs versus tumor cells to peptide cytotoxicity are discussed. Finally, compared with structurally idealized amphiphilic α‐helical peptides, non‐idealized peptides can possess higher tumoricidal activity, but are less hemolytic and less susceptible to serum inhibition.
The rational design of highly stable, amphiphilic helical peptides
Authors: CHMIELELE'SKI JEAN , LIPTON MARK
Published: August 2010

A computer algorithm was devised for the evaluation of helical stability of potentially amphiphilic peptide sequences of specified length containing a set number of leucines in the hydrophobic region. All possible combinations of Glu, Lys and Gln in the hydrophilic region are rated using a set of empirical rules for salt bridge formation in α‐helices, and the sequences which rate the highest are displayed. The rules for salt bridge formation were largely derived from published studies on the effects of salt bridges on helical stability. The algorithm was tested by redesigning a known amphiphilic α‐helical peptide, αlB or 1, which has been shown to aggregate into four‐helix bundles. Comparison of the circular dichroism spectra of two peptides, 2 and 3, to 1 demonstrated that the redesigned peptide with the highest priority score from the algorithm, 2, was more helical when aggregated and slightly more helical as a monomer, whereas the peptide with the low priority score, 3, was somewhat less helical when aggregated and much less helical when monomeric. These results support the design of the algorithm, although conclusions based on aggregation data are complicated by the importance of interhelix contacts in the bundle. Further studies are underway to examine the reliability of the algorithm's predictions regarding the design of other helical peptides.
Solid‐phase peptide synthesis and biological activity of bovine thymopoietin II (bTP–11)
Authors: SMITH D. DAVID , CONLON J. MICHAEL , PETZEL JANIS , CHEN LEI , MURPHY RICHARD F. , MORLEY BARBARA J.
Published: August 2010

Bovine thymopoietin (bTP), a 49 amino acid polypeptide, was synthesized using Merrifield's solid‐phase peptide synthesis methodology. The polypeptide was purified using anion‐exchange chromatography and reversed‐phase HPLC and characterized by mass spectrometry and amino acid analysis of the full‐length peptide and of products derived from digestion withStaphylococcus aureusV8 protease. The biological activity of the synthesized product was tested in several assay systems. Synthetic bTP was found to induce the expression of Thy 1.2 antigen on T‐lymphocytes from athymic mice, in agreement with previous studies on the biological activity of endogenous bTP. Biological activity at skeletal muscle and neuronal nicotinic acetylcholine receptor sites, as reported by others for bTP, could not be confirmed in our studies. The absence of biological activity at nicotinic receptor sites may be related to the results of a recent report demonstrating the presence of a cobratoxin‐like molecule in preparations of natural bTP. These data indicate that synthetic peptides have an important role for the evaluation of the specificity of the biological activity of polypeptides.
Further studies into the Boc/solid‐phase synthesis of Ser(P)‐ and Thr(P)‐containing peptides
Authors: PERICH JOHN W. , TERZI EVELYNE , CARNAZZI ERIC , SEYER RENE , TRIFILIEFF ELISABETH
Published: August 2010

The Ser(P)‐containing peptide corresponding to phospholamban 11‐19, Ac‐Ala‐Ile‐Arg‐Are‐Ala‐Ser(P)‐Thr‐Ile‐Glu‐NH2, was prepared by the use of Boc‐Ser(PO3Ph2)‐OH in Boc/solid‐phase peptide synthesis followed by HF cleavage of the peptide from the polystyrene resin and subsequent platinum‐mediated hydrogenolytic cleavage of the phenyl phosphate groups. A study of the HF deprotection step showed that extensive dephosphorylation of the Ser(PO3Ph2)‐residue occurred using three commonly used HF conditions and gave rise to large quantities of the Ser‐containing peptide. The subsequent study of model peptide systems under standard HF conditions established firstly that the extent of dephosphorylation was dependent on the HF‐contact time, and secondly that the Ser(PO3Ph2) residue underwent dephosphorylation at a slightly higher rate than the Thr(PO3Ph2) residue. © Munksgaard 1994.
Coupling constants and hydrogen bonds as experimental restraints in a distance geometry refinement protocol *
Authors: MIERKE DALE F. , GEYER ARMIN , KESSLER HORST
Published: August 2010

A refinement procedure commonly used after distance geometry calculations has been modified to include the use of experimental restraints from coupling constants and hydrogen bonds. Fewer experimental distance constraints (NOEs) are available for peptides as compared to proteins; therefore it is important to incorporate other conformational restraints into refinement methods. The procedure was applied to a cyclic hexapeptide containing two thioamide substitutions, cyclo(‐Glyl‐Pro2‐Phe3ψ[CS‐NH]Va14‐D‐Phe5‐Phe6ψ[CS‐NH]‐). Distance geometry was used to study this peptide, since no potential energy parameters, required in molecular mechanics or dynamics calculations, are available for the thioamide. This is a general problem in the study of peptidomimetics; physicochemical properties of heteroatoms are required within a self‐consistent force field. Here, we illustrate the use of metric matrix distance geometry followed by refinement with distance and angle driven dynamics (DADD). We also introduce a new way to handle intramolecular hydrogen bonds by an additional very small and flexible restraint. This method is a viable alternative for the conformational examination of peptides and peptidomimetics. The modifications described here should also find use in the conformational determination of flexible regions of proteins, where the number of NOEs are limited. © Munksgaard 1994.
In vitro cross‐linking of calf lens α‐crystallin by malondialdehyde
Authors: LIBONDI TEODOSIO , RAGONE RAFFAELE , VINCENTI DONATELLA , STIUSO PAOLA , AURICCHIO GIACINTO , COLONNA GIOVANNI
Published: August 2010

The effect of malondialdehyde on structural features of bovine x‐crystallin has been investigated by absorption and fluorescence spectroscopy as well as by far‐UV circular dichroism. Experimental evidence suggests the occurrence of intermolecular cross‐linking induced by malondialdehyde. This cross‐linking does not seem to affect the tryptophan environment, as suggested by intrinsic protein fluorescence. On the contrary, the time dependence of far‐UV dichroic activity indicates that the cross‐linking is accompanied by a secondary structure change. The formation of high molecular mass aggregates, evidenced by electrophoresis in denaturing conditions, leads to irreversible x‐crystallin aggregation due to extensive intermolecular cross‐linking. Since malondialdehyde is produced in vivo as a breakdown product of lipid peroxidation, the possible involvement of this molecule in the pathological mechanism of cataract formation has been briefly discussed. © Munksgaard 1994.
Turn induction by N‐aminoproline Comparison of the Gly‐Pro‐Gly and Glyψ[CO‐NH‐N]Pro‐Gly sequences
Authors: ZERKOUT S. , DUPONT V. , AUBRY A. , VIDAL J. , COLLET A. , VICHERAT A. , MARRAUD M.
Published: August 2010

The folded structure induced by the N‐aminoproline residue (the hydrazino analogue of proline, denoted hPro) in the Boc‐Gly1‐hPro2‐Gly3‐NHiPr hydrazino tripeptide has been characterized in the solid state by X‐ray diffraction, and compared to the usual βII‐turn structure in the Boc‐Gly1‐Pro2‐Gly36‐NHiPr cognate tripeptide. It is stabilized by a bifurcated hydrogen bond in which (Gly3)NH interacts with both (Gly1)CO and (hPro2)Nx. This conformation is retained in CH2Cl2 and CHC13 solutions, and allows an overall folded conformation of the hydrazino tripeptide in which (iPr)NH is hydrogen‐bonded to (Boc)CO. The hPro α‐hydrazino acid residue appears to promote a local folded structure, and might behave as a β‐turn mimic. © Munksgaard 1994.
Computational molecular modeling in peptide drug design
Authors: NIKIFOROVICH GREGORY V.
Published: August 2010

The review concentrates on practical applications of computer molecular modeling in peptide drug design. The examples of the predictions (successful or not) made by computational modeling before synthesis of peptide analogs, not the explanations provided after synthesis and biological testing of peptides, are discussed. The review spans over 20 years of predictions made by computer molecular modeling for bradykinin, angio‐tensin, thyrotropin‐releasing factor, tuftsin, substance P, CCK‐related peptides, luliberin, α‐melanotropin and opioid peptides. The described examples are discussed in terms of finding the optimal way to use computer modeling for peptide design. The step‐by‐step‘technology’of peptide design is outlined in detail. © Munksgaard 1994.
Monitoring the progress of non‐enzymatic glycation in vitro
Authors: SHAW SEAN M. , CRABBE M. JAMES C.
Published: August 2010

The progress of in vitro non‐enzymatic glycation of bovine serum albumin was followed by using 14C‐glucose and a nitroblue tetrazolium assay, absorption and fluorescence spectroscopy, SDS gel electrophoresis and protease digestion. The number of adducts detectable using both 14C‐tracers and a fructosamine assay remained low at physiological glucose concentrations, fewer than five adducts being detectable. When glucose concentrations > 1.0 M were used the number of adducts was found to greatly exceed the number of lysyl residues available in BSA, indicative of cross‐linking between Maillard products. Incubation of BSA with glucose concentrations of up to 160 mM for one month produced no observable increase in molecular weight by SDS gel electrophoresis, showing that at physiological glucose concentrations, increases in molecular weight were minimal for short incubation periods, any marked changes (indicated by non‐penetration of the 7.5% SDS gel) requiring nine months incubation with ≥ 20 mM glucose. Increases in absorption were proportional to both the glucose concentration and the incubation time. Several absorption peaks, at 370, 488 and 554 nm, were consistent in appearance throughout the course of each incubation. Fluorescence spectroscopy of the modified proteins showed a disappearance of the fluorescence associated with peptide bonds and aromatic residues and the appearance of a broad peak at longer wavelengths due to the wide range of absorptive/fluorescent wavelengths of the developing Maillard products. Protease digestion gave similar patterns with non‐glycated and glycated protein, suggesting that glycation did not block digestion sites, and that partial digestion did not cause significant further exposure of susceptible sites. Our results show that while glycation ultimately results in protein conformational changes and the formation of large molecular weight species, these occur at a relatively late stage in the maturation of protein Maillard products, after ≥ nine months of incubation with glucose concentrations of ≥ 20 mM. Monitoring of AGE maturation in itro is better accomplished by following incorporation of 14C‐[UL]‐glucose and spectroscopic techniques than by monitoring of molecular weight changes. © Munksgaard 1994.
Methanol‐mediated, modular self‐assembly of antiparallel β‐dimers
Authors: KARLE ISABELLA K. , RANGANATHAN DARSHAN
Published: August 2010

The backbone‐modified tripeptide Bz‐Aib‐NHCOCO‐Aib‐OMe (l), in which the central Cα has been replaced with CO, self‐assembles in the solid state into highly ordered two‐dimensional arrays through MeOH mediated intermolecular stacking of dimeric ‘disk’ modules formed by an antiparallel β‐sheet‐type arrangement of tripeptide molecules. The Cα(C′O)N(C′O) (C′O)NCα segment is nearly coplanar (average deviation from a mean least‐square plane is ± 0.037 Å). The oxalyl moiety forms two pseudo‐C5 intramolecular hydrogen bonds. Crystal parameters are as follows: C18H23N3O6CH3OH, triclinic space group P1 a = 9.796(4), b = 10.348(3), c = 11.836(4) Å, α= 78.28(3), β= 73.72(3), γ= 69.45(3), R = 0.082 for 1747 reflections measured with ‖Fo‖3σ(F).
A topographical model of μ‐opioid and brain somatostatin receptor selective ligands NMR and molecular dynamics studies
Authors: KAZMIFRSKI WIESLAW M. , FERGUSON R.D. , LIPKOWSKI ANDRZEJ W. , HRUBY VICTOR J.
Published: August 2010

We have refined the1H NMR‐based conformations of the μ‐opioid receptor selective peptides related to somatostatin of general formula Xxx‐Yyy1‐Cys‐Zzz‐D‐Trp‐Lys(Orn)5‐Thr‐Peh‐Thr8‐NH2, where Xxx, Yyy, Zzz are 0,d‐Phe and Tyr for 1; 0,d‐Tic and Tyr for 2; Gly,d‐Tic and Tyr for 3; and 0,d‐Phe and Tic for 4, respectively, (Kazmierski et al.,J. Am. Chem.113, 2275–2283), using a molecular‐dynamics approach. We present evidence that the NMR data are compatible with βII′‐, γ‐ and γ′‐turns for the central tetrapeptide Tyr‐D‐Trp‐Lys/Orn‐Thr. Based on detailed structural and topographical considerations, we suggest that the μ‐opioid receptor selectivity of 2 is due to a particular spatial arrangement of aromatic side chains ofd‐Tic1and Tyr3(7.5 Å), and that the opioid receptor recognition domain is located in the N‐terminal part of the peptide while the somatostatin receptor recognition domain is determined by the central, turn forming part of this class of cyclic peptides. A model for a μ‐opioid selective ligand has emerged from these studies that shows excellent structural similarities to rigid opioid alkaloids.
‘Tethered ligand’derived pentapeptide agonists of thrombin receptor: a study of side chain requirements for human platelet activation and GTPase stimulation
Authors: NATARAJAN SESHA , RIEXINGER DOUGLAS , PELUSO MARIANNE , SEILER STEVEN M.
Published: August 2010

Proteolytic action of α‐thrombin on human thrombin receptor results in cleavage of a portion of the N‐terminus, thereby generating a‘tethered ligand’at the newly exposed N‐terminus, which then activates the receptor in an intramolecular fashion. Agonist peptides incorporating the amino acid sequence of the newly exposed N‐terminal portion of the cleaved receptor cause receptor activation without requiring prior cleavage of the receptor by thrombin. The pentapeptide amide Ser‐Phe‐Leu‐Leu‐Arg‐NH2, which retains the N‐terminal sequence of the‘tethered ligand’of the receptor, has been shown to be the minimum sequence to cause receptor activation. To understand the importance of the side chains of various residues within the pentapeptide amide, we carried out an extensive structure‐activity study of the ability of peptides to stimulate gel‐filtered platelet aggregation. In this study 106 pentapeptide amides were synthesized, utilizing naturally occurring l‐amino acids, unnatural amino acids, D‐amino acids and N‐methyl amino acids for replacements. At position‐1, charged residues (acidic or basic) were not tolerated, and the size and shape of the residue were important. Position‐2 tolerated only aromatic residues. Position‐3 accommodated various residues. A significant finding of this study was that two very different residues, [3‐(2‐naphthyl)]‐l‐alanine and l‐arginine, when substituted for leucine residue at position‐3, resulted in more active agonists. At position‐4 aromatic and aliphatic residues were well tolerated, whereas basic and acidic residues were less tolerated. Position‐5 mimicked position‐3 in its ability to tolerate a wide range of residues. In general, an acidic residue was not preferred in any position. In all positions a ‐Phe‐residue was tolerated. Positions 1 and 3 tolerated ‐Pro‐residue, whereas positions 2, 4 and 5 did not. d‐Amino acid substitution was not tolerated in any position. While N‐methylation of residues at positions‐3 and ‐4 led to poor agonists, at position‐2 it resulted in an inactive analog. These studies define the side chain requirements for the‘tethered ligand’derived pentapeptide agonists of thrombin receptor for human platelet activation.

Selected peptides were tested for ability to stimulate platelet membrane GTPase. A good correlation was observed between peptides that stimulate GTPase and those that stimulate platelet aggregation, with the general finding that 3‐10‐fold higher concentrations were required to half maximally activate GTPase when compared with the concentrations needed for activation of platelet aggregation. © Munksgaard 1995.
Role of peptide hydrophilicity on determination of microsequencing efficiency
Authors: CHANG NAN‐SHAN
Published: August 2010

The successful sequencing of short peptides on hydrophobic polyvinylidene difluoride membrane (PVDF‐P) has been problematic. In this study the sequencing efficiency of various short synthetic peptides on charged‐modified PVDF (PVDF‐N) and chemically treated glass‐fiber membranes or discs has been examined. These modified membranes provided better repetitive yields or sequencing efficiency than the unmodified PVDF‐P. In contrast, there were no significant differences among the resulting initial yields for all the tested membranes, indicating that the modified membranes did not interfere with the coupling/cleaving reactions. Methanol at 1% increased the solubility of phenylisothiocyanate (PITC) in heptane for gas‐phase delivery during the coupling reaction, whereas this addition of methanol failed to increase the coupling efficiency. Reduction of chemical background noise by replacing triethylamine (TEA) with diisopropylethylamine (DIPEA) also failed to increase the coupling efficiency. Polybrene strengthened the peptide binding to both PVDF‐P and PVDF‐N, but increased the amount of carry‐over of PTH‐amino acid from the current cycle to the next. Nonetheless, hydrophilic peptides had higher sequencing recoveries and repetitive yields than hydrophobic peptides when sequenced on all the tested membranes. This relationship was further verified by testing a synthetic peptide with decreasing hydrophilicity by sequential deletions of 2 amino acid residues from its N‐terminus. A decreasing sequencing efficiency was observed, which correlated with the reduced hydrophilicity and peptide length. Similar results were obtained when testing peptide fragments with decreasing hydrophilicity by deletions of amino acids from the C‐terminus. The physical properties of peptides determine their binding stability with supporting membranes and the ease with which they are extracted by sequencing chemicals, thus affecting in part the sequencing efficiency. © Munksgaard 1995.
Use of the 3‐nitro‐2‐pyridine sulfenyl protecting group to introduce Nε‐branching at lysine during solid‐phase peptide synthesis I. Application to the synthesis of a peptide template containing two addressable sites
Authors: RAJAGOPALAN SHYAMALA , HECK THOMAS J. , IWAMOTO TAKEO , TOMICH JOHN M.
Published: August 2010

TASPs (template‐assembled synthetic peptides) are generated by the covalent attachment of linear peptides to a common peptide backbone, thus generating larger synthetic peptides/proteins with prefolded structure. In this work we present a strategy for the synthesis of a heterotemplate‐assembled synthetic peptide containing two addressable sites. This orthogonal protection strategy would allow the selective introduction of different peptide chains via the ε‐amino functions of template lysines being protected by either fluorenylmethoxycarbonyl (Fmoc) or 3‐nitro‐2‐pyridine sulfenyl (Npys) groups. The Nα‐Boc‐Nε‐Npys‐l‐lysine required for solid‐phase peptide synthesis (SPPS) is not readily available at a reasonable cost. To facilitate the more widespread use of this reagent we have compared the two published procedures for synthesizing this protected amino acid and evaluated the suitability of the products for SPPS. Two resin‐bound peptides, a tripeptide Ac‐G‐K‐Npys)‐G‐resin and an octapeptide template Ac‐P1‐K2‐K3‐L4‐Ks‐K6‐P7‐G8‐resin, were synthesized by SPPS. The ε‐amino functions of lysines K2 & K6 and K3 & K5 of the octapeptide were protected by Fmoc and Npys groups, respectively. Secondly, these peptides were used to evaluate various reagents and reaction conditions for the deprotection of the ε‐amino function of lysines bearing the Nε‐Npys protecting group. A procedure for the optimized selective and quantitative deprotection of the Npys group from the ε‐amino function of lysine in a resin‐bound peptide using 2‐mercaptopyridine‐N‐oxide is described. © Munksgaard 1995.
Stability of subtilisins and related proteinases (subtilases)
Authors: GENOV NICOLAY , FILIPPI BRUNO , DOLASHKA PAVLINA , WILSON KEITH S. , BETZEL CHRISTIAN
Published: August 2010

The stability towards thermal and chemical (guanidine hydrochloride, GnHCl) denaturation of six inhibited subtilases (mesentericopeptidase, subtilisins BPN′, Carlsberg and DY, proteinase K and thermitase) has been investigated by kinetic and equilibrium studies. The unfolding processes were monitored by circular dichroic and fluorescence spectroscopy. Experiments in the absence and presence of extraneous calcium in the concentration range 2×10−3‐10−1 M were performed. The presence of calcium in the weak calcium binding site changes the denaturation drastically. The heat‐ (or GnHCl‐) induced unfolding curves obtained using CD spectroscopy show two independent transitions which seem not to have been resolved before. The presence of Ca2+ in the second (third in the case of thermitase) binding site increases the Tm, values by 11‐21 °C and the δGD(H2O) values obtained from denaturation experiments in GnHCl by 6.7‐7.2 kcal/mol when an extraneous Ca2+ concentration of 2 × 10−2 M was used. One interpretation is that the initial step of denaturation in the presence of added calcium is the formation of a partially unfolded intermediate form, retaining a highly ordered structure with 60‐85% of the a‐helix structure of the native enzyme. This intermediate then unfolds at a temperature considerably higher than that of the same proteinases in the absence of added Ca2+. The free energy of stabilization of the intermediates is increased by 1.8‐2.8 times in comparison with that for the unfolding reactions of the subtilases with empty Ca2/Ca3 binding sites. A second interpretation is that the two steps in the unfolding curves correspond to enzyme without and with calcium in the weak binding site. Fluorescence experiments confirm the mechanism involving the formation of intermediate states. The results are discussed in relation to the X‐ray models of the six subtilases.