The primary structure of the β-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa

The complete amino acid sequence of the β-subunit of protocatechuate 3,4-dioxygenase was determined. The β-subunit contained four methionine residues. Thus, five peptides were obtained after cleavage of the carboxymethylated β-subunit with cyanogen bromide, and were isolated on Sephadex G-75 column chromatography. The amino acid sequences of the cyanogen bromide peptides were established by characterization of the peptides obtained after digestion with trypsin, chymotrypsin, thermolysin, or Staphylococcus aureus protease. The major sequencing techniques used were automated and manual Edman degradations. The five cyanogen bromide peptides were aligned by means of the amino acid sequences of the peptides containing methionine purified from the tryptic hydrolysate of the carboxymethylated β-subunit. The amino acid sequence of all the 238 residues was as follows: ProAlaGlnAspAsnSerArgPheValIleArgAsp ArgAsnTrpHis ProLysAlaLeuThrPro-Asp — TyrLysThrSerIleAlaArg SerProArgGlnAla LeuValSerIleProGlnSer — IleSerGluThrThrGly ProAsnPheSerHisLeu GlyPheGlyAlaHisAsp-His — AspLeuLeuLeuAsnPheAsn AsnGlyGlyLeu ProIleGlyGluArgIle-Ile — ValAlaGlyArgValValAsp GlnTyrGlyLysPro ValProAsnThrLeuValGluMet — TrpGlnAlaAsnAla GlyGlyArgTyrArg HisLysAsnAspArgTyrLeuAlaPro — LeuAspProAsn PheGlyGlyValGly ArgCysLeuThrAspSerAspGlyTyrTyr — SerPheArg ThrIleLysProGlyPro TyrProTrpArgAsnGlyProAsnAsp — TrpArgProAla HisIleHisPheGlyIle SerGlyProSerIleAlaThr-Lys — LeuIleThrGlnLeuTyr PheGluGlyAspPro LeuIleProMetCysProIleVal — LysSerIleAlaAsn ProGluAlaValGlnGln LeuIleAlaLysLeuAspMetAsnAsn — AlaAsnProMet AsnCysLeuAlaTyr ArgPheAspIleValLeuArgGlyGlnArgLysThrHis PheGluAsnCys. The sequence published earlier in summary form (Iwaki et al., 1979, J. Biochem.86, 1159–1162) contained a few errors which are pointed out in this paper.     

Amino acid sequence of a protease inhibitor isolated from Sarcophaga bullata determined by mass spectrometry.

The amino acid sequence of a protease inhibitor isolated from the hemolymph of Sarcophaga bullata larvae was determined by tandem mass spectrometry. Homology considerations with respect to other protease inhibitors with known primary structures assisted in the choice of the procedure followed in the sequence determination and in the alignment of the various peptides obtained from specific chemical cleavage at cysteines and enzyme digests of the S. bullata protease inhibitor. The resulting sequence of 57 residues is as follows: Val Asp Lys Ser Ala Cys Leu Gln Pro Lys Glu Val Gly Pro Cys Arg Lys Ser Asp Phe Val Phe Phe Tyr Asn Ala Asp Thr Lys Ala Cys Glu Glu Phe Leu Tyr Gly Gly Cys Arg Gly Asn Asp Asn Arg Phe Asn Thr Lys Glu Glu Cys Glu Lys Leu Cys Leu.     

Carbon‐11 radiolabeling of an oligopeptide containing tryptophan hydrochloride via a Pictet‐Spengler reaction using carbon‐11 formaldehyde

A procedure for the synthesis of a¹¹C‐labeled oligopeptide containing [1‐¹¹C]1,2,3,4‐tetrahydro‐β‐carboline‐3‐carboxylic acid ([1‐¹¹C]Tpi) from the corresponding Trp?HCl‐containing peptides has been developed involving a Pictet‐Spengler reaction with [¹¹C]formaldehyde. The synthesis of [1‐¹¹C]Tpi from Trp and [¹¹C]formaldehyde was examined as a model reaction with the aim of developing a facile and effective method for the labeling of peptides with carbon‐11. The Pictet‐Spengler reaction of Trp and [¹¹C]formaldehyde in acidic media (TsOH or HCl) afforded the desired [1‐¹¹C]Tpi in a moderate radiochemical yield. Herein, the application of a Pictet‐Spengler reaction to an aqueous solution of Trp?HCl gave the desired product with a radiochemical yield of 45.2%. The RGD peptide cyclo[Arg‐Gly‐Asp‐D‐Tyr‐Lys] was then selected as a substrate for the labeling reaction with [¹¹C]formaldehyde. The radiolabeling of a Trp?HCl‐containing RGD peptide using the Pictet‐Spengler reaction was successful. Furthermore, the remote‐controlled synthesis of a [1‐¹¹C]Tpi‐containing RGD peptide was attempted by using an automatic production system to generate [¹¹C]CH₃I. The radiochemical yield of the [1‐¹¹C]Tpi‐containing RGD at the end of synthesis (EOS) was 5.9 ± 1.9% (n = 4), for a total synthesis time of about 35 min. The specific activity was 85.7 ± 9.4 GBq/µmol at the EOS. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit.

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.     

Sequences of tryptic peptides containing the five cysteinyl residues of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Tryptic peptides which account for all five cysteinyl residues in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have been purified and sequenced. Collectively, these peptides contain 94 of the approximately 500 amino acid residues per molecule of subunit. Due to one incomplete cleavage at a site for trypsin and two incomplete chymotryptic-like cleavages, eight major radioactive peptides (rather than five as predicted) were recovered from tryptic digests of the enzyme that had been carboxymethylated with [³H]iodoacetate. The established sequences are: GlyTyrThrAlaPheValHisCys¹Lys TyrValAspLeuAlaLeuLysGluGluAspLeuIleAla GlyGlyGluHisValLeuCys¹AlaTyr AlaGlyTyrGlyTyrValAlaThrAlaAlaHisPheAla AlaGluSerSerThrGlyThrAspValGluValCys¹ ThrThrAsxAsxPheThrArg AlaCys¹ThrProIleIleSerGlyGlyMetAsnAla LeuArg ProPheAlaGluAlaCys¹HisAlaPheTrpLeuGly GlyAsnPheIleLys In these peptides, radioactive carboxymethylcysteinyl residues are denoted with asterisks and the sites of incomplete cleavage with vertical wavy lines. None of the peptides appear homologous with either of two cysteinyl-containing, active-site peptides previously isolated from spinach ribulosebisphosphate carboxylase/oxygenase.     

Angiotensin II restricted analogs with biological activity in the erythrocytic cycle of Plasmodium falciparum

The anti‐plasmodial activity of conformationally restricted analogs of angiotensin II against Plasmodium gallinaceum has been described. To observe activity against another Plasmodium species, invasion of red blood cells by Plasmodium falciparum was analyzed. Analogs restricted with lactam or disulfide bridges were synthesized to determine their effects and constraints in the peptide–parasite interaction. The analogs were synthesized using tert‐butoxycarbonyl and fluoromethoxycarbonyl solid phase methods, purified by liquid chromatography, and characterized by mass spectrometry. Results indicated that the lactam bridge restricted analogs 1 (Glu‐Asp‐Arg‐Orn ‐Val‐Tyr‐Ile‐His‐Pro‐Phe) and 3 (Asp‐Glu‐Arg‐Val‐Orn ‐Tyr‐Ile‐His‐Pro‐Phe) showed activity toward inhibition of ring formation stage of P. falciparum erythrocytic cycle, preventing invasion in about 40% of the erythrocytes. The disulfide‐bridged analog 10 (Cys‐Asp‐Arg‐Cys ‐Val‐Tyr‐Ile‐His‐Pro‐Phe) was less effective yet significant, showing a 25% decrease in infection of new erythrocytes. In all cases, the peptides presented no pressor activity, and hydrophobic interactions between the aromatic and alkyl amino acid side chains were preserved, a factor proven important in efficacy against P. gallinaceum. In contrast, hydrophilic interactions between the Asp¹ carboxyl and Arg² guanidyl groups proved not to be as important as they were in the case of P. gallinaceum, while interactions between the Arg² guanidyl and Tyr⁴ hydroxyl groups were not important in either case. The β‐turn conformation was predominant in all of the active peptides, proving importance in anti‐plasmodial activity. This approach provides insight for understanding the importance of each amino acid residue on the native angiotensin II structure and a new direction for the design of potential chemotherapeutic agents. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Analogues of deltorphin I containing conformationally restricted amino acids in position 2: structure and opioid activity

New analogues of deltorphin I (DT I, Tyr‐d ‐Ala‐Phe‐Asp‐Val‐Val‐Gly‐NH₂), with the d ‐Ala residue in position 2 replaced by α‐methyl‐β‐azido(amino, 1‐pyrrolidinyl, 1‐piperidinyl or 4‐morpholinyl)alanine, were synthesized by a combination of solid‐phase and solution methods. All ten new analogues were tested for receptor affinity and selectivity to μ‐ and δ‐opioid receptors. The affinity of analogues containing (R) or (S)‐α‐methyl‐β‐azidoalanine in position 2 to δ‐receptors strongly depended on the chirality of the α,α‐disubstituted residue. Peptide II , containing (S)‐α‐methyl‐β‐azidoalanine in position 2, displayed excellent δ‐receptor selectivity with its δ‐receptor affinity being only three times lower than that of DT I. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

β‐Amino acids containing peptides and click‐cyclized peptide as β‐turn mimics: a comparative study with ‘conventional’ lactam‐ and disulfide‐bridged hexapeptides

The increasing interest in click chemistry and its use to stabilize turn structures led us to compare the propensity for β‐turn stabilization of different analogs designed as mimics of the β‐turn structure found in tendamistat. The β‐turn conformation of linear β‐amino acid‐containing peptides and triazole‐cyclized analogs were compared to ‘conventional’ lactam‐ and disulfide‐bridged hexapeptide analogs. Their 3D structures and their propensity to fold in β‐turns in solution, and for those not structured in solution in the presence of α‐amylase, were analyzed by NMR spectroscopy and by restrained molecular dynamics with energy minimization. The linear tetrapeptide Ac‐Ser‐Trp‐Arg‐Tyr‐NH₂ and both the amide bond‐cyclized, c[Pro‐Ser‐Trp‐Arg‐Tyr‐D ‐Ala] and the disulfide‐bridged, Ac‐c[Cys‐Ser‐Trp‐Arg‐Tyr‐Cys]‐NH₂ hexapeptides adopt dominantly in solution a β‐turn conformation closely related to the one observed in tendamistat. On the contrary, the β‐amino acid‐containing peptides such as Ac‐(R)‐β³‐hSer‐(S)‐Trp‐(S)‐β³‐hArg‐(S)‐β³‐hTyr‐NH₂, and the triazole cyclic peptide, c[Lys‐Ser‐Trp‐Arg‐Tyr‐βtA]‐NH₂, both specifically designed to mimic this β‐turn, do not adopt stable structures in solution and do not show any characteristics of β‐turn conformation. However, these unstructured peptides specifically interact in the active site of α‐amylase, as shown by TrNOESY and saturation transfer difference NMR experiments performed in the presence of the enzyme, and are displaced by acarbose, a specific α‐amylase inhibitor. Thus, in contrast to amide‐cyclized or disulfide‐bridged hexapeptides, β‐amino acid‐containing peptides and click‐cyclized peptides may not be regarded as β‐turn stabilizers, but can be considered as potential β‐turn inducers. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Detailed characterization of an anti-factor IX monoclonal antibody that neutralizes the prolonged ox brain prothrombin time of hemophilia B(M) by synthetic peptides

In a previous study, we prepared a monoclonal antibody (MoAb) to coagulation factor IX (FIX), designated 65-10, which interfered with the activation of FIX by the activated factor XI/Ca(2+) and neutralized the prolonged ox brain prothrombin time of hemophilia B(M) [11,12]. The location of the epitope on the FIX for 65-10 MoAb is (168) Ile-Thr-Gln-Ser-Thr-Gln-Ser-Phe-Asn-Asp-Phe-Thr-Arg-Val-Val(182) [21]. In this paper, we studied in more detail an epitope on FIX using the systematic substitution of different amino acids at each residue of the epitope peptides and the influence of the epitope peptide on the prolonged ox brain prothrombin time of the hemophilia B(M) plasma of 65-10 MoAb. In the replacement set of amino acids, peptides showing low or no reactivity to 65-10 were (175)Phe --> Asp, Glu, Gly, Lys, Arg, Thr, Val, (176)Asn --> Asp, Glu, Phe, Ile, Lys, Leu, Pro, Val, Tyr, (177)Asp --> Cys, Glu, Phe, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr, and (178) Phe --> Pro. These results imply that a hydrophobic molecule of (175) Phe, a hydrophilic molecule of (176)Asn, and a negative charge molecule of (177)Asp were important to the epitope. The 65-10 MoAb antibody neutralized the prolonged ox brain prothrombin time of hemophilia B(M) Nagoya 2 ((180)Arg -->Trp) and Kashihara ((181)Val --> Phe) as well as B(M) Kiryu ((313)Val --> Asp) and Niigata ((390)Ala --> Val). This reaction was inhibited by preincubation with a (168) Ile-Thr-Gln-Ser-Thr-Gln-Ser-Phe-Asn-Asp-Phe-Thr-Arg-Val-Val(182) peptide conjugated with bovine serum albumin (BSA). 65-10 MoAb that has been useful in detailing epitopes will be useful for qualitative analysis of hemophilia B(M).     

A fluorescently labeled undecapeptide derived from a protein in royal jelly of the honeybee—royalisin—for specific detection of oxidized low‐density lipoprotein

The probes for detection of oxidized low‐density lipoprotein (ox‐LDL) in plasma and in atherosclerotic plaques are expected to facilitate the diagnosis, prevention, and treatment of atherosclerosis. Recently, we have reported that a heptapeptide (Lys‐Trp‐Tyr‐Lys‐Asp‐Gly‐Asp, KP6) coupled through the ε‐amino group of N‐terminal Lys to fluorescein isothiocyanate (FITC), (FITC)KP6, can be useful as a fluorescent probe for specific detection of ox‐LDL. In the present study, to develop a novel fluorescent peptide for specific detection of ox‐LDL, we investigated the interaction (with ox‐LDL) of an undecapeptide corresponding to positions 41 to 51 of a potent antimicrobial protein (royalisin, which consists of 51 residues; from royal jelly of honeybees), conjugated at the N‐terminus to FITC in the presence of 6‐amino‐n‐caproic acid (AC) linker, (FITC‐AC)‐royalisin P11, which contains both sequences, Phe‐Lys‐Asp and Asp‐Lys‐Tyr, similar to Tyr‐Lys‐Asp in (FITC)KP6. The (FITC‐AC)‐royalisin P11 bound with high specificity to ox‐LDL in a dose‐dependent manner, through the binding to major lipid components in ox‐LDL (lysophosphatidylcholine and oxidized phosphatidylcholine). In contrast, a (FITC‐AC)‐shuffled royalisin P11 peptide, in which sequences Phe‐Lys‐Asp and Asp‐Lys‐Tyr were modified to Lys‐Phe‐Asp and Asp‐Tyr‐Lys, respectively, hardly bound to LDL and ox‐LDL. These findings strongly suggest that (FITC‐AC)‐royalisin P11 may be an effective fluorescent probe for specific detection of ox‐LDL and that royalisin from the royal jelly of honeybees may play a role in the treatment of atherosclerosis through the specific binding of the region at positions 41 to 51 to ox‐LDL.     

NH2-terminal sequences of DNA-, heparin-, and gelatin-binding tryptic fragments from human plasma fibronectin

NH₂-terminal sequence analysis was performed on subregions of human plasma fibronectin including 24,000-dalton (24K) DNA-binding, 29,000-dalton (29K) gelatin-binding, and 18,000-dalton (18K) heparin-binding tryptic fragments. These fragments were obtained from fibronectin after extensive trypsin digestion followed by sequential affinity purification on gelatin-Sepharose, heparin-agarose, and DNA-cellulose columns. The gelatin-binding fragment was further purified by gel filtration on Sephadex G-100, and the DNA-binding and heparin-binding fragments were further purified by high-performance liquid chromatography. The 29K fragment had the following NH₂-terminal sequence: AlaAlaValTyrGlnProGlnProHisProGlnProPro (Pro)TyrGlyHis HisValThrAsp(His)(Thr)ValValTyrGly(Ser) ?(Ser)?-Lys. The NH₂-terminal sequence of a 50K, gelatin-binding, subtilisin fragment by L. I. Gold, A. Garcia-Pardo, B. Prangione, E. C. Franklin, and E. Pearlstein (1979, Proc. Nat. Acad. Sci. USA76, 4803–4807) is identical to positions 3–19 (with the exception of some ambiguity at position 14) of the 29K fragment. These data strongly suggest that the 29K tryptic fragment is included in the 50K subtilisin fragment, and that subtilisin cleaves fibronectin between the Ala²Val³ residues of the 29K tryptic fragment. The 18K heparin-binding fragment had the following NH₂-terminal sequence: (Glu)AlaProGlnProHisCysIleSerLysTyrIle LeuTyrTrpAspProLysAsnSerValGly?(Pro) LysGluAla?(Val)(Pro). The 29K gelatin-binding and 18K heparin-binding fragments have proline-rich NH₂-terminal sequences suggesting that they may have arisen from protease-sensitive, random coil regions of fibronectin corresponding to interdomain regions preceding macromolecular-binding domains. Both of these fragments contain the identical sequence ProGlnProHis, a sequence which may be repeated in other interdomain regions of fibronectin. The 24K DNA-binding fragment has the following NH₂-terminal sequence: SerAspThrValProSerProCysAspLeuGlnPhe ValGluValThrAspVal LysValThrIleMetTrpThrProProGluSerAla ValThrGlyTyrArgVal AspValCysProValAsnLeuProGlyGluHisGly Gln(Cys)LeuProIleSer. The sequence of positions 9–22 are homologous to positions 15–28 of the α chain of DNA-dependent RNA polymerase from Escherichia coli. The homology observed suggests that this stretch of amino acids may be a DNA-binding site.     

Design of peptidase‐resistant peptide inhibitors of myosin light chain kinase

Myosin light chain kinase (MLCK) is a key regulator of various forms of cell motility including smooth muscle contraction, cell migration, cytokinesis, receptor capping, secretion, etc. Inhibition of MLCK activity in endothelial and epithelial monolayers using cell‐permeant peptide Arg‐Lys‐Lys‐Tyr‐Lys‐Tyr‐Arg‐Arg‐Lys (PIK, P eptide I nhibitor of K inase) allows protecting the barrier capacity, suggesting a potential medical use of PIK. However, low stability of L ‐PIK in a biological milieu prompts for development of more stable L ‐PIK analogues for use as experimental tools in basic and drug‐oriented biomedical research. Previously, we designed PIK1, H‐(N^αMe)Arg‐Lys‐Lys‐Tyr‐Lys‐Tyr‐Arg‐Arg‐Lys‐NH₂, that was 2.5‐fold more resistant to peptidases in human plasma in vitro than L ‐PIK and equal to it as MLCK inhibitor. In order to further enhance proteolytic stability of PIK inhibitor, we designed the set of six site‐protected peptides based on L ‐PIK and PIK1 degradation patterns in human plasma as revealed by ¹H‐NMR analysis. Implemented modifications increased half‐live of the PIK‐related peptides in plasma about 10‐fold, and these compounds retained 25–100% of L ‐PIK inhibitory activity toward MLCK in vitro. Based on stability and functional activity ranking, PIK2, H‐(N^αMe)Arg‐Lys‐Lys‐Tyr‐Lys‐Tyr‐Arg‐D ‐Arg‐Lys‐NH₂, was identified as the most stable and effective L ‐PIK analogue. PIK2 was able to decrease myosin light chain phosphorylation in endothelial cells stimulated with thrombin, and this effect correlated with the inhibition by PIK2 of thrombin‐induced endothelial hyperpermeability in vitro. Therefore, PIK2 could be used as novel alternative to other cell‐permeant inhibitors of MLCK in cell culture‐based and in vivo studies where MLCK catalytic activity inhibition is required. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Bioactive conformations of two seminal delta opioid receptor penta‐peptides inferred from free‐energy profiles

Delta‐opioid (DOP) receptors are members of the G protein‐coupled receptor (GPCR) sub‐family of opioid receptors, and are evolutionarily related, with homology exceeding 70%, to cognate mu‐opioid (MOP), kappa‐opioid (KOP), and nociceptin opioid (NOP) receptors. DOP receptors are considered attractive drug targets for pain management because agonists at these receptors are reported to exhibit strong antinociceptive activity with relatively few side effects. Among the most potent analgesics targeting the DOP receptor are the linear and cyclic enkephalin analogs known as DADLE (Tyr‐D ‐Ala‐Gly‐Phe‐D ‐Leu) and DPDPE (Tyr‐D ‐Pen‐Gly‐Phe‐D ‐Pen), respectively. Several computational and experimental studies have been carried out over the years to characterize the conformational profile of these penta‐peptides with the ultimate goal of designing potent peptidomimetic agonists for the DOP receptor. The computational studies published to date, however, have investigated only a limited range of timescales and used over‐simplified representations of the solvent environment. We provide here a thorough exploration of the conformational space of DADLE and DPDPE in an explicit solvent, using microsecond‐scale molecular dynamics and bias‐exchange metadynamics simulations. Free‐energy profiles derived from these simulations point to a small number of DADLE and DPDPE conformational minima in solution, which are separated by relatively small energy barriers. Candidate bioactive forms of these peptides are selected from identified common spatial arrangements of key pharmacophoric points within all sampled conformations. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 21–27, 2014.     

Interaction of Met297 in the seventh transmembrane segment of the tachykinin NK2 receptor with neurokinin A

We report the use of thiol chemistry to define specific and reversible disulfide interactions of Cys-substituted NK2 receptor mutants with analogues of neurokinin A (NKA) containing single cysteine substitutions. The NKA analogues were N-biotinylated to facilitate the rapid detection of covalent analogue-receptor interactions utilizing streptavidin reactivity. N-biotinyl-[Tyr1,Cys9]NKA, N-biotinyl-[Tyr1,Cys10]NKA were both found to reversibly disulfide bond to the NK2 receptor mutant Met297 --> Cys. This is consistent with the improved affinities of these particular analogues for the Met297 --> Cys receptor as compared with those for the wild-type and Met297 --> Leu receptors. In our three-dimensional model, Met297 occupies the equivalent position in helix 7 to the retinal binding Lys296 in rhodopsin. Binding of the NK2 receptor antagonist [3H]SR 48968 and of 125I-NKA was used to characterize additional receptor mutants. It seems that the aromatic residues Trp99 (helix 3), His198 (helix 5), Tyr266, His267, and Phe270 play an important role in NKA binding as structural determinants. The existence of overlapping SR 48968 and NKA binding sites is also evident. These data suggest that the peptide binding site of the NK2R is at least in part formed by residues buried deep within the transmembrane bundle and that this intramembranous binding domain may correspond to the binding sites for substantially smaller endogenous GPCR ligands.     

Effects of orally administrated amino acids on myofibrillar proteolysis in chicks

We examined the effects of orally administrated amino acids on myfibrillar proteolysis in food-deprived chicks. Plasma N(tau)-methylhistidine concentration, as an index of myofibrillar proteolysis, was decreased by the administration of Glu, Gly, Ala, Leu, Ile, Ser, Thr, Met, Trp, Asn, Gln, Pro, Lys and Arg but not by Asp, Val, Phe, Tyr or His to chicks. Orally administrated Cys was fatal to chicks. These results indicate that oral Glu, Gly, Ala, Leu, Ile, Ser, Thr, Met, Trp, Asn, Gln, Pro, Lys and Arg administration suppressed myofibrillar proteolysis in chicks.     

New t‐butyl based aspartate protecting groups preventing aspartimide formation in Fmoc SPPS

Obtaining homogenous aspartyl‐containing peptides via Fmoc/tBu chemistry is often an insurmountable obstacle. A generic solution for this issue utilising an optimised side‐chain protection strategy that minimises aspartimide formation would therefore be most desirable. To this end, we developed the following new derivatives: Fmoc‐Asp(OEpe)‐OH (Epe = 3‐ethyl‐3‐pentyl), Fmoc‐Asp(OPhp)‐OH (Php = 4‐n‐propyl‐4‐heptyl) and Fmoc‐Asp(OBno)‐OH (Bno = 5‐n‐butyl‐5‐nonyl). We have compared their effectiveness against that of Fmoc‐Asp(OtBu)‐OH and Fmoc‐Asp(OMpe)‐OH in the well‐established scorpion toxin II model peptide variants H‐Val‐Lys‐Asp‐Asn/Arg‐Tyr‐Ile‐OH by treatments of the peptidyl resins with the Fmoc removal reagents containing piperidine and DBU at both room and elevated temperatures. The new derivatives proved to be extremely effective in minimising aspartimide by‐products in each application. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Characteristic of aromatic amino acid substitution at alpha 96 of hemoglobin

Replacement of valine by tryptophan or tyrosine at position alpha96 of the alpha chain (alpha96Val), located in the alpha(1)beta(2) subunit interface of hemoglobin leads to low oxygen affinity hemoglobin, and has been suggested to be due to the extra stability introduced by an aromatic amino acid at the alpha96 position. The characteristic of aromatic amino acid substitution at the alpha96 of hemoglobin has been further investigated by producing double mutant r Hb (alpha42Tyr --> Phe, alpha96Val --> Trp). r Hb (alpha42Tyr --> Phe) is known to exhibit almost no cooperativity in binding oxygen, and possesses high oxygen affinity due to the disruption of the hydrogen bond between alpha42Tyr and beta99Asp in thealpha(1)beta(2) subunit interface of deoxy Hb A. The second mutation, alpha96Val -->Trp, may compensate the functional defects of r Hb (alpha42Tyr --> Phe), if the stability due to the introduction of trypophan at the alpha 96 position is strong enough to overcome the defect of r Hb (alpha42Tyr --> Phe). Double mutant r Hb (alpha42Tyr --> Phe, alpha96Val --> Trp) exhibited almost no cooperativity in binding oxygen and possessed high oxygen affinity, similarly to that of r Hb (alpha42Tyr --> Phe). (1)H NMR spectroscopic data of r Hb (alpha42Tyr --> Phe, alpha96Val --> Trp) also showed a very unstable deoxy-quaternary structure. The present investigation has demonstrated that the presence of the crucible hydrogen bond between alpha 42Tyr and beta 99Asp is essential for the novel oxygen binding properties of deoxy Hb (alpha96Val --> Trp) .     

Lysozyme modification by the fenton reaction and gamma radiation

A comparative study was performed on lysozyme modification after exposure to Fenton reagent (Fe(II)/H2 O2) or hydroxyl radicals produced by y radiation. The conditions were adjusted to obtain, with both systems, a 50% loss of activity of the modified ensemble. Gamma radiation modified almost all types of amino acid residues in the enzyme, with little specificity. The modification order was Tyr > Met = Cys > Lys > Ile + Leu > Gly > Pro = Phe > Thr + Ala > Trp = Ser > Arg > Asp + Glu, with 42 mol of modified residues per initial mole of native enzyme. In contrast, when the enzyme was exposed to the Fenton reaction, only some types of amino acids were modified. Furthermore, a smaller number of residues (13.5) were damaged per initial mole of enzyme. The order of the modified residues was Tyr > Cys > Trp > Met His > Ile + Leu > Val > Arg. These results demonstrate that the modifications elicited by these two free radical sources follow different mechanisms. An intramolecular free radical chain reaction is proposed to play a dominant role in the oxidative modification of the protein promoted by gamma radiation.     

The primary structure of the COOH-terminal half of cholera toxin subunit A1 containing the ADP-ribosylation site

The sequence of 96 amino acid residues from the COOH-terminus of the active subunit of cholera toxin, A₁, has been determined as PheAsnValAsnAspVal LeuGlyAlaTyrAlaProHisProAsxGluGlu GluValSerAlaLeuGlyGly IleProTyrSerGluIleTyrGlyTrpTyrArg ValHisPheGlyValLeuAsp GluGluLeuHisArgGlyTyrArgAspArgTyr TyrSerAsnLeuAspIleAla ProAlaAlaAspGlyTyrGlyLeuAlaGlyPhe ProProGluHisArgAlaTrp ArgGluGluProTrpIleHisHisAlaPro ProGlyCysGlyAsnAlaProArg(OH). This is the largest fragment obtained by BrCN cleavage of the subunit A₁ (M_r 23,000), and has previously been indicated to contain the active site for the adenylate cyclase-stimulating activity. Unequivocal identification of the COOH-terminal structure was achieved by separation and analysis of the terminal peptide after the specific chemical cleavage at the only cysteine residue in A₁ polypeptide. The site of self ADP-ribosylation in the A₁ subunit [C. Y. Lai, Q.-C. Xia, and P. T. Salotra (1983) Biochem. Biophys. Res. Commun.116, 341–348] has now been identified as Arg-50 of this peptide, 46 residues removed from the COOH-terminus. The cysteine that forms disulfide bridge to A₂ subunit in the holotoxin is at position 91.     

Molecular Modeling of the NPY Binding Site on the Y1 Receptor

A three-dimensional model of the neuropeptide Y (NPY) - rat Y₁ (rY₁) receptor complex and of the NPY _13-36 - rY₁ receptor complex was constructed by molecular modeling based on the electron density projection map of rhodopsin and on site-directed mutagenesis studies of neuropeptide receptors. In order to further guide the modeling, the nucleotide sequences encoding Trp287, Cys295 and His297 in the third extracellular loop of the rY₁ receptor, were altered by site-directed mutagenesis experiments. Single-point mutated receptors were expressed in COS-7 cells, and tested for their ability to bind radio labelled NPY (³H-NPY). Mutations of Trp287 and His297 completely abolished binding of ³H-NPY. The Cys295Ser mutation only slightly decreased the binding of ³H-NPY, suggesting that the involvement of Cys295 in a disulphide bond is not essential for maintaining the correct three-dimensional structure of the binding site for NPY. Molecular dynamics simulations of NPY-rY₁ receptor interactions suggested that Asp199, Asp103 and Asp286 in the receptor interact, respectively, with Lys4, Arg33 and Arg35 of NPY. The simulations also suggested that His297 acts as a hydrogen acceptor from Arg35 in NPY, and that Tyr1 of NPY interacts with a binding pocket on the receptor formed by Asn115, Asp286, Trp287 and His297. Tyr36 in NPY interacted both with Thr41 and Tyr99 via hydrogen bonds, and also with Asn296, His297 and Phe301. The present study suggests that amino acid residues at the extracellular end of the transmembrane helices and in the extracellular loops are strongly involved in binding to NPY and NPY_13-36.Electronic Supplementary Material available.