Tyr Pro Research Articles

Human immunodeficiency viral protease is catalytically active as a fusion protein: Characterization of the fusion and native enzymes produced in Escherichia coli

Processing of the gag and pol gene precursor proteins of retroviruses is essential for the production of mature infectious virions. The processing is directed by a viral protease that itself is part of these precursors and is presumed to cleave itself autocatalytically. To facilitate study of this process, the protease was produced as a fusion protein in Escherichia coli. In this construct, the 10,793-Da protease was preceeded by two copies of a modified IgG binding domain derived from protein A. The IgG binding domain was linked to the protease by an AspPro peptide bond which could not be cleaved by the viral protease. A dimer of the 25,400-Da fusion protein was catalytically active, specifically cleaving a substrate peptide at the correct TyrPro bond. Thus, the fusion protein could serve as a model of the viral gag- pol polyprotein. The finding that the fusion protein was catalytically active supports the suggestion that a gag- pol dimer can initiate a proteolytic cascade after budding of the immature virus. The fusion protein also provided a source of authentic protease. The protease was released from the fusion construct by incubation with formic acid, cleaving the AspPro linkage which had been inserted between the IgG binding domain and the protease.

Opioid Activities of Morphiceptin Analogs Derived from Human β-Casein

Abstract Four tetrapeptide amides with the N-terminal Tyr–Pro sequence were synthesized as possible opioid agonists that can be produced by degradation of human β-casein. When these peptides were tested for their ability to bind to the μ and δ opioid receptors in rat brain, only H–Tyr–Pro–Phe–Val-NH2 was active, showing the 60% increased affinity for the μ receptors as compared with morphiceptin (H–Tyr–Pro–Phe–Pro–NH2) derived from bovine β-casein. It was highly μ-selective, as well as morphiceptin, with the μ/δ-selectivity ratio of 285. Other three analogs, H–Tyr–Pro–Ser–Phe–NH2, H–Tyr–Pro–Val–Arg–NH2 and H–Tyr–Pro–Val–Pro–NH2, were almost completely inactive. These results suggested that, for the morphiceptin-like tetrapeptide amides, the presence of Phe at position 3 is essential to elicit an activity to bind to the μ opioid receptors. Conformational considerations by measuring the CD spectra indicated that the sequence of tetrapeptide amide Tyr–Pro–Xxx–Pro(or Val)–NH2 is an important structural requirement to interact with the μ opioid receptors.

Novel Fluorogenic Substrates for Assaying Retroviral Proteases by Resonance Energy Transfer

The 11-kD protease (PR) encoded by the human immunodeficiency virus 1 (HIV-1) is essential for the correct processing of viral polyproteins and the maturation of infectious virus, and is therefore a target for the design of selective acquired immunodeficiency syndrome (AIDS) therapeutics. To facilitate the identification of novel inhibitors of HIV-1 PR, as well as to permit detailed studies on the enzymology and inhibition of this enzyme, a continuous assay for its activity was developed that was based on intramolecular fluorescence resonance energy transfer (RET). The assay used the quenched fluorogenic substrate 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL)--Ser Gln Asn Tyr Pro Ile Val Gln--5-[(2-aminoethyl)amino]naphthalene-1 sulfonic acid (EDANS), whose peptide sequence is derived from a natural processing site for HIV-1 PR. Incubation of recombinant HIV-1 PR with the fluorogenic substrate resulted in specific cleavage at the Tyr-Pro bond and a time-dependent increase in fluorescence intensity that was linearly related to the extent of substrate hydrolysis. An internally quenched fluorogenic substrate was also designed that was selectively cleaved by the related PR from avian myeloblastosis virus (AMV). The fluorescence quantum yields of the HIV-1 PR and AMV PR substrates in the RET assay increased by 40.0- and 34.4-fold, respectively, per mole of substrate cleaved. Because of its simplicity, rapidity, and precision in the determination of reaction rates required for kinetic analysis, this method offers many advantages over the commonly used high-performance liquid chromatography- or electrophoresis-based assays for peptide substrate hydrolysis by retroviral PRs.

Polyproline, β‐turn helices. Novel secondary structures proposed for the tandem repeats within rhodopsin, synaptophysin, synexin, gliadin, RNA polymerase II, hordein, and gluten

Seven proteins each contain 8 to 52 tandem repeats of a unique class of oligopeptide. The consensus peptide for each is rhodopsin Tyr Pro Pro Gln Gly synaptophysin Tyr Gly Pro Gln Gly synexin Tyr Pro Pro Pro Pro Gly gliadin Tyr Pro Pro Pro Gln Pro RNA polymerase II Tyr Ser Pro Thr Ser Pro Ser hordein Phe Pro Gln Gln Pro Gln Gln Pro gluten Tyr Pro Thr Ser Pro Gln Gln Gly Tyr Although there is obvious variation of sequence and of length, the penta- to nonapeptides share an initial Tyr (or Phe) and have high Pro contents and abundant Gly, Gln, and Ser. We have evaluated helical models that both recognize the uniqueness of these sequence repeats and accommodate variations on the basic theme. We have developed a group of related helical models for these proteins with about three oligopeptide repeats per turn of 10-20 A. These models share several common features: Most of the phi dihedral angles are -54 degrees, to accommodate Pro at all positions except the first (Tyr). Except for the beta-turns, most psi dihedral angles are near +140 degrees as found in polyproline. Each oligopeptide has at least one beta-turn; several have two. Some contain a cis-Tyr, Pro peptide bond; a few have a cis-bond plus one beta-turn. Tyr side chains vary from totally exposed to buried within the helices and could move to accommodate either external hydrophobic interactions or phosphorylation. The several related structures seem to be readily interconverted without major change in the overall helical parameters, and therein may lie the key to their functions.

A rapid purification method for rat liver pyruvate carboxylase and amino acid sequence analyses of NH 2-terminal and biotin peptide

A rapid method for the purification of pyruvate carboxylase from rat liver has been developed. The method involves extraction of the enzyme from frozen liver powder followed by polyethylene glycol fractionation and avidin-affinity chromatography. The purified enzyme has a specific activity of 9–10 μmol/min/mg protein when assayed at 22 °C in the presence of acetyl-CoA. Polyacrylamide gel electrophoresis of the preparation in the presence of sodium dodecyl sulfate showed the presence of one protein band with an estimated M r 125,000 and no significant contamination by other biotin-containing enzymes. In addition to being rapid, the method is advantageous because prior isolation of mitochondria is not necessary. Using these preparations we have determined the sequence of the first 15 amino acids from the NH 2-terminal end of the molecule to be SerGlyProValAlaPro LeuAsnValLeuLeuLeuGluTyrPro. The sequence of the 24 amino acid residues around the biotin site was determined to be GlyAlaProLeuValLeuSerAlaMet -biocytinMetGluThr ValValThrSerProThrGluGlyThrIleArg.

Xenopus homeobox-containing cDNAs expressed in early development.

We report the isolation of six different homeobox-containing genes in Xenopus laevis which are expressed during early embryogenesis. cDNA clones of all of them were partially sequenced including two from previously isolated Xenopus genomic loci and found to contain homeodomains which share a high degree of homology with the Antennapedia protein (50 to 59 out of 60 amino acids). We find a short region of homology (consensus Ile Tyr Pro Trp Met) in four of the cDNAs, which is also present in Antennapedia and that may correspond to a bend region preceding the homeodomain. Northern blots have been performed to show the transcriptional pattern through early frog embryogenesis. Three of the genes are expressed only during a very narrow period of embryogenesis, reinforcing the view that homeobox genes are developmentally regulated.

Ser 7]neurotensin: isolation from guinea pig intestine

Using three antisera to neurotensin of defined regional specificity, a novel neurotensin has been identified in extracts of guinea pig brain and small intestine. The primary structure of the peptide was established as: pGlu Leu Tyr GIu Asn Lys Ser Arg Arg Pro Tyr He Leu. Guinea pig neurotensin differs from bovine neurotensin by substitution of a prolyl residue by a seryl residue at position 7. Synthetic [Ser 7]neurotensin showed identical Chromatographie and immunochemical properties to guinea pig neurotensin. This difference in primary structure may account for some of the anomalous pharmacological effects of bovine neurotensin on guinea pig tissues.

A toxic thionin from Pyrularia pubera: Purification, properties, and amino acid sequence

A low-molecular-weight cytotoxic protein has been purified from Pyrularia pubera Michx. (Santalaceae). By comparison with the behavior of proteins of known molecular weight during Sephadex G-75 gel filtration and denaturing electrophoresis, a molecular weight of somewhat less than 6000 is indicated. Purification involves ammonium sulfate fractionation followed by either gel filtration on Sephadex G-75 or separation on a carboxymethyl cellulose CM52 column. At concentrations of 0.04 mg/ml the protein causes visible disruption of cultured mouse B16 melanoma cells. The complete amino acid sequence has been determined. The toxin contains 47 amino acids arranged as follows: LysSerCysCysArgAsnThrTrpAlaArgAsnCys TyrAsnValCys ArgLeuProGlyThrIleSerArgGluIleCysAlaLys LysCysAspCysLys IleIleSerGlyThrThrCysProSerAspTyrProLys OH. The protein is clearly a thionin, as shown by its close resemblance to the thionins from wheat and barley, to the viscotoxins from mistletoes, and to crambin.

Proton magnetic resonance studies on dermorphin and its peptide fragments.

AbstractDermorphin (TyrD‐AlaPheGlyTyrProSerNH2), a potent natural peptide opioid, its synthetic L‐Ala2 analog, and all the N fragments from the tripeptide (TyrD‐AlaPheNH2) to the parent hexapeptide amide were characterized for the first time by means of proton nmr spectroscopy at 11.74 T. Assignments of most protons of dermorphin were facilitated by the study of the N‐terminal fragments. Comparison of spectroscopic parameters with relative pharmacological activity is proposed as a possible means of studying flexible agonists in solution.

The primary structure of the COOH-terminal half of cholera toxin subunit A 1 containing the ADP-ribosylation site

The sequence of 96 amino acid residues from the COOH-terminus of the active subunit of cholera toxin, A 1, 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 A 1 ( 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 A 1 polypeptide. The site of self ADP-ribosylation in the A 1 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 A 2 subunit in the holotoxin is at position 91.

ChemInform Abstract: STUDIES OF BITTER PEPTIDES FROM CASEIN HYDROLYZATE. VI. SYNTHESES AND BITTER TASTE OF BPIC (VAL‐TYR‐PRO‐PHE‐PRO‐PRO‐GLY‐ILE‐ASN‐HIS) AND ITS ANALOGS AND FRAGMENTS

In order to investigate the relationship between chemical structure and bitter taste, the bitter peptide BPIc (Val–Tyr–Pro–Phe–Pro–Pro–Gly–Ile–Asn–His) isolated from casein hydrolyzate by Minamiura et al. and its analogs and fragments were synthesized. BPIc, whose threshold value of bitter taste was 0.05 mM, was found to be one of the most bitter compounds, like quinine and phenylthiourea. However, [Gly5,6]- and [Gly9,10]-BPIc, and N-terminal octa- and heptapeptide fragments of BPIc possessed much weaker bitterness than BPIc. The results suggested that 5,6-proline and the basic nature of C-terminal are necessary for the strong bitterness exhibited by BPIc.

ChemInform Abstract: CHEMICAL SYNTHESIS OF A HEAT‐STABLE ENTEROTOXIN PRODUCED BY ENTEROTOXIGENIC ESCHERICHIA COLI STRAIN 18D

AbstractFür das hitzebeständige Enterotoxin aus dem ente rotoxigenen Ecoli‐Stamm 18D sind früher die Strukturen (IVa) bzw. (IVb) vorgeschlagen worden.

ChemInform Abstract: STUDIES OF BITTER PEPTIDES FROM CASEIN HYDROLYZATE. VII. BITTERNESS OF THE RETRO‐BPIA (VAL‐ILE‐PHE‐PRO‐PRO‐GLY‐ARG) AND ITS FRAGMENTS

AbstractDas Peptid (IIa) ("retro‐BPIa"), das die umgekehrte Aminosäurebaustein‐Sequenz wie das extrem bittere BPIa (I) aufweist, sowie die Fragmente (IIb), (III), (IV) und (V) werden (durch schrittweisen Aufbau nach üblicher Methodik) synthetisiert.

Amino acid sequence of the K-peptide generated by limited proteolysis of glucose dehydrogenase from Bacillus megaterium by proteinase K

Proteolysis of glucose dehydrogenase from Bacillus megaterium with proteinase K apparently generated two fragments. The small fragment, designated as K-peptide, was sequenced and its covalent structure was determined as SerSerGluAlaSerTyrValThrGlyIleThrLeuPhe AlaAspGlyGlyMetThrGlnTyrProSerPheGluAla GlyArgGly. The sequence analysis showed that the K-peptide consists of two identical fragments, one of which is lacking one serine residue at the aminoterminus.

Studies of Bitter Peptides from Casein Hydrolyzate. VII. Bitterness of the retro-BPIa (Val–Ile–Phe–Pro–Pro–Gly–Arg) and Its Fragments

Abstract To explain the bitter taste exhibited by BPIa (Arg–Gly–Pro–Pro–Phe–Ile–Val) which was isolated from casein hydrolyzate, we have proposed the following requirement: its characteristic spatial structure: the basic moiety in the N-terminal and the hydrophobic moiety in the C-terminal were affected to each other by prolylprolyl residue, is necessary for the bitterness to be exhibited. As for BPIc (Val–Tyr–Pro–Phe–Pro–Pro–Gly–Ile–Asn-His), which is the other fraction, although it exhibited as strong and bitter a taste as BPIa, the basic moiety of BPIc is located in the C-terminal and its hydrophobic moiety is located in the N-terminal. The authors synthesized retro-BPIa with the reverse peptide sequence and its fragments. The retro-BPIa exhibited as strong and bitter a taste as BPIa. However, the bitterness of the fragments of retro-BPIa was far weaker than that of retro-BPIa.

Chemical Synthesis of a Heat-stable Enterotoxin Produced by Enterotoxigenic Escherichia coli Strain 18D

Abstract Two peptides with the two primary structures of 18 amino acid residues proposed for a heat-stable enterotoxin from enterotoxigenic Escherichia coli strain 18D were synthesized by solution methods and their physicochemical and biological properties were compared with those of native toxin. One of these peptides (Asn–Thr–Phe–Tyr–Cys–Cys–Glu–Leu–Cys–Cys–Asn–Pro–Ala–Cys–Ala–Gly–Cys–Tyr) showed the same heat-stability and 1H-NMR spectrum as those of the native toxin and evoked fluid secretion in suckling mice at a dose of 1.5–2.0 ng, which is similar to the effective dose of native toxin. Moreover, its toxicity was neutralized by antisera against the native toxin. The other peptide (Asn–Thr–Phe–Tyr–Cys–Cys–Glu–Leu–Cys–Cys–Tyr–Pro–Ala–Cys–Ala–Gly–Cys–Asn) showed different physicochemical and biological behaviors from those of the native toxin and exhibited toxic activity at a dose of 50–80 ng, which is about thirty times the effective dose of the native toxin. Moreover, its toxicity was not neutralized by antisera against the native toxin. These observations indicate that the former peptide is identical to native toxin but that the latter peptide differs in properties from the native toxin.

Studies of Bitter Peptides from Casein Hydrolyzate. VI. Syntheses and Bitter Taste of BPIc (Val–Tyr–Pro–Phe–Pro–Pro–Gly–Ile–Asn–His) and Its Analogs and Fragments

Abstract In order to investigate the relationship between chemical structure and bitter taste, the bitter peptide BPIc (Val–Tyr–Pro–Phe–Pro–Pro–Gly–Ile–Asn–His) isolated from casein hydrolyzate by Minamiura et al. and its analogs and fragments were synthesized. BPIc, whose threshold value of bitter taste was 0.05 mM, was found to be one of the most bitter compounds, like quinine and phenylthiourea. However, [Gly5,6]- and [Gly9,10]-BPIc, and N-terminal octa- and heptapeptide fragments of BPIc possessed much weaker bitterness than BPIc. The results suggested that 5,6-proline and the basic nature of C-terminal are necessary for the strong bitterness exhibited by BPIc.

Interaction of metal ions with peptide hormones

Among our studies concerning the coordination chemistry of naturally occurring peptides we have been studying recently metal ion interaction with two hypothalamic hormones: thyroliberin (TRF) and melanostatin (MIF). These two hormones are the only tripeptides among all the pituitary hormones described until now [1–4]. TRF (PyrHisProNH 2) and MIF (ProLeuGlyNH 2) have been extensively studied in various biological aspects [5], among others their possible use in the clinical treatment of metal diseases [6]. Many hormone analogues have been synthesized in order to solve the problem of the relation between biological activity and chemical structure [7]. In most papers the critical role of the histidine residue in TRF biological activity is suggested. Our main interest concerns the coordination abilities of both hormones and some of their analogues and the relation between the binding modes and the ligand conformation (structure). Spectroscopic (NMR, EPR, absorption and CD spectra) and potentiometric techniques were used to solve these problems. Though the MIF binding mode to Cu(II) and Ni(II) ions looks classical, the strongly basic Pro nitrogen donor on the N-terminal leads to the formation of very low concentration of 110 (1N) complex species [8]. The histidine residue of TRF ligand is a major binding site of metal ions. The other two residues however, have a critical importance for the complex equilibria and the structure of the formed species [9, 10]. The replacement of pyroglutamic acid with picolinic acid in the TRF molecule causes a major change in the structure of its Cu(II) and Ni(II) complexes. E.g., in the Cu(II)PicHis system a very specific equilibrium between dimeric and monomeric species was observed [11]. The introduction of tyrosine residue in the TRF sequence in place of histidine also changes the peptide coordination ability. In the Ni(II)PyrTyrProNH 2 system a direct involvement of ProNH 2 residue in the metal binding was proposed [11]. TRF analogue with nicotinic acid in place of pyroglutamic acid forms very unstable Cu(II) complexes due to the unfavourable meta-position of the nitrogen donor with respect to carbonyl in the pyridine ring. Also the TRF analogue with ProNHNH 2 residue in the C-terminal position forms much weaker Cu(II) and Ni(II) complexes than the TRF peptide. The studies which were done for both hormones show that naturally occurring peptides may form relatively strong complexes with some metal ions and their chemical and physical properties may be very specific for each ligand. The latter conclusion becomes evident when the coordination abilities of the different chemical analogues ( e.g. of TRF) are compared.

Spectroscopic and potentiometric studies on coordination abilities of tetrapeptides containing proline and tyrosine or phenylalanine

The insertion of proline residues, a ‘break-point’ [1–3] in the second position of a tetrapeptide divides the ligand molecule into two fragments which are potentially able to interact with metal ions independently, i.e. the N-terminal aminoacid ( e.g. via NH 2, CO) and two C-terminal aminoacid residues ( via e.g. 2N −, COO −). The introduction of tyrosine to such a peptide sequence complicates solution equilibria distinctly [1, 3]. Phenylalanine, which is not able to use its side-chain for the direct metal ion binding, is a good analogue for tyrosine and both residues were used for the synthesis and the study of the coordination abilities of tetrapeptides. The spectroscopic and thermodynamic studies of cupric complexes with tetrapeptides: GlyProGlyTyr, GlyProTyrGly, TyrProGlyGly, GlyProPheGly, GlyProDPheGly and PheProGlyGly have shown that the metal peptide interaction always starts at the N-terminal (NH 2, CO donors). The tyrosine residue may involve its side-chain OH in the metal ion binding with formation of monomeric or dimeric species. The complex equilibria strongly depend on the position of tyrosine residue in the peptide sequence. The lack of the direct involvement of phenylalanine side-chain in the direct metal ion coordination leads to much simpler equilibria. The presence of proline residue in the ligand molecule causes very unusual coordination modes in the formal complex species with formation of unusually large chelate rings. The combination of spectroscopic and potentiometric approaches led to the satisfactory description of very complicated systems which may be used as reasonable spectroscopic and structural models for biosystems.

Structure-function relationship of [2Fe2S] ferredoxins and design of a model molecule

[2Fe2S] ferredoxins isolated from various plants and algae comprise 93–99 amino acid residues and resemble each other not only in sequences, but also in physiological functions. One of them isolated from Spirulina platensis was subjected to X-ray analysis and its three dimensional structure is now known. [2Fe2S] ferredoxins of a different type are found in halobacteria and comprise 128 amino acid residues. Both types of the [2Fe2S] ferredoxins exhibit low redox potentials. By comparing the amino acid sequences of 28 [2Fe2S] ferredoxins and the tertiary structure of S. platensis ferredoxin we predicted a common three-dimensional structure to the [2Fe2S] ferredoxins and proposed a molecular surface area to be interacting with FNR. An artificial small molecule composed of 20 amino acid residues is designed on the basis of the tertiary structure of S. platensis ferredoxin. The amino acid sequence was predicted to be ProTyrSerCysArgAlaGlyAlaCysSerThrCysAlaGly ProLeuLeuThr CysVal which should have a [2Fe2S] cluster with a low redox potential