Peptide synthesis using o-nitrophenylsulfenyl N-carboxy alpha-amino acid anhydrides. Sequential oligopeptides having alternate gamma-methyl L-glutamyl and L-phenylalanyl residues.

A series of sequential oligopeptides having the sequence alternating γ-methyl L -glutamyl and L -phenylalanyl residues have been successfully prepared by a rapid method involving the reaction of o-nitrophenylsulfenyl N-carboxy α-amino acid anhydrides with amino acid and peptide esters. The sequential oligopeptides, which are interesting from a conformational aspect, were obtained in optical pure forms above 74% yields. This result demonstrates that the o-nitrophenylsulfenyl N-carboxy α-amino acid anhydride method is especially useful for easy synthesis of protected oligopeptides with o-nitrophenylsulfenyl group.     

Aminopeptidase N from Streptococcus salivarius subsp. thermophilus NCDO 573: purification and properties

A 96 kDa aminopeptidase was purified from Streptococcus salivarius subsp. thermophilus NCDO 573. The enzyme had similar properties to aminopeptidases isolated from lactococci and lactobacilli and showed a high degree of N -terminal amino acid sequence homology to aminopeptidase N from Lactococcus lactis subsp. cremoris. It catalysed the hydrolysis of a range of aminoacyl 4-nitroanilides and 7-amido-4-methylcoumarin derivatives, dipeptides, tripeptides and oligopeptides. In common with aminopeptidases from other lactic acid bacteria, the enzyme from Strep. salivarius subsp. thermophilus showed highest activity with lysyl derivatives but was also very active with arginyl and leucyl derivatives. Relative activity with alanyl, phenylalanyl, tyrosyl, seryl and valyl derivatives was considerably lower and with glycyl, glutamyl and prolyl derivatives almost negligible. The aminopeptidase also catalysed the hydrolysis of dipeptides and tripeptides but mostly at rates much less than that with L-lysyl-4-nitroanilide and oligopeptides. The enzyme catalysed the successive hydrolysis of various amino acid residues from the N -terminus of several oligopeptides but it was unable to cleave peptide bonds on the N -terminal side of a proline residue.     

Fragmentomics of proteins and natural oligopeptides

The term fragmentomics is grounded and defined. Theoretical structure-function analysis of all possible fragments of a protein molecule was performed under the concept of fragmentomics to determine the regions that could be potential sources of regulatory oligopeptides. For this purpose, we used the data on the primary structure of bovine hemoglobin, the information contained in the EROP-Moscow database on the structures and functions of natural oligopeptides, and a specialized software package. This analysis revealed natural nonhemoglobin oligopeptides containing hemoglobin fragments and natural oligopeptides with the structure precisely coinciding with hemoglobin fragments. The most abundant of them are neuropeptides, antimicrobial oligopeptides, and hormones. It was demonstrated that the tetrapeptide and larger fragments of hemoglobin identified in nonhemoglobin oligopeptides and possessing a mentioned activity are present in the amino acid sequences of experimentally determined hemoglobin oligopeptides with the same function. The proposed approach allowed us to discover new potentially active sites in the hemoglobin amino acid sequence not yet studied experimentally. The possibility of natural formation of regulatory oligopeptides from hemoglobin molecules and other food proteins is discussed, as well as the generation of an exogenous oligopeptide pool in the gastrointestinal tract, and how the results match the concept of natural continuum of regulatory oligopeptides.     

Convenient solid-phase synthesis of oligopeptides using pentacoordinated phosphoranes with amino acid residue as building blocks

The reactive intermediates of pentacoordinated phosphoranes with amino acids (P(5)-AA) as building blocks, which were obtained by the reaction of O-phenylene phosphorochloridate with N,O-bis(trimethylsilyl)amino acids, were linked to a solid-phase support containing a hydroxymethyl polystyrene functional group. The first amino acid residue was coupled to the solid-phase support after washing the resin with organic solvent. Repeating the procedure led to oligopeptides linked on the resin. A series of free oligopeptides including tetra-Gly, di-Val, tri-Val, di-Leu, di-Phe, and Phe-Leu were obtained after cleavage from solid-phase support. The structure of these oligopeptides were determined by IR, (1)H NMR, FAB-MS, and HPLC.     

Conformational studies of alanine oligopeptides by nuclear magnetic resonance.

We have studied the nmr spectra of the series of alanine oligopeptides containing a methoxyethoxyethoxyacetyl blocking group on the N-terminal residue and a morpholino blocking group on the C-terminal residue. Spectra were measured in chloroform–trifluoroacetic acid solvent systems. For oligomers with chain lengths of five or more, “double peaks” are observed for the α-CH protons. Addition of trifluoroacetic acid causes the peaks to coalesce. The amount of trifluoroacetic acid necessary for coalescence increases from the pentamer to the nonamer. These findings are general since alanine oligomers with different blocking groups exhibit similar “double peak” phenomena. We explain the “double peak” phenomenon in terms of specific folded forms of the oligopeptides which arise from intramolecular hydrogen bonding. Additional evidence for such hydrogen bonding is presented based upon infrared studies. Slight aggregation probably occurs for the pentamer and hexamer which may stabilize the folded forms.     

Aminopeptidases in webbing clothes moth larvae. properties and specificities of enzymes of highest electrophoretic mobility.

The group of aminopeptidase bands from Tineola bisselliella larvae with highest electrophoretic mobility in polyacrylamide gels were purified further and partially separated by ion exchange chromatography. Three aminopeptidase bands were present in this material and were very similar with respect to their pH optima (7-7), their molecular weight of 94,000, their responses to metal ions and enzyme inhibitors and in their substrate specificity requirements. Kinetic constants were obtained for the hydrolysis of 17 different alpha-aminoacyl-beta-naphthylamides by these aminopeptidases, the most favoured substrates being the derivatives of alanine, methionine, proline, leucine, glycine, glutamic acid, lysine and arginine. The enzymes also hydrolyse amino acid amides, dipeptides, dipeptide amides, tripeptides and oligopeptides at the N-terminal end. These enzymes differ from the other aminopeptides in T. bisselliella in being able to hydrolyse bonds involving proline.     

Study of the relationship between conformation and nature of side chains in linear oligopeptides: homologous series derived from β-branched amino acid residues

Conformational analysts of the three homologous series, from dipeptide to heptapeptide of monodisperse, N- and C-protected oligopeptides derived from the β-branched α-amino acid residues L-valine, L-isoleucine, and D-allo-isoleucine is reported. Occurrence of intermolecular β conformations in the higher oligopeptides in the solid state was established by means of infrared absorption. The extent of intramolecularly hydrogen-bonded folded structure formation was assessed as a function of chain length in solvents of low polarity at high dilution. Statistical coil and intermolecular β-conformations were shown to exist in alcohols and aqueous alcoholic mixtures. The results obtained indicate that, branching positions being equal, configuration of the asymmetric carbon atom in the lateral chain, overall bulkiness and lyophobic character of the amino acid side chains are all important factors in determining the stability of the ordered conformations of oligopeptides.     

Antimicrobial and other oligopeptides of grapes

Structures and functions of about 700 oligopeptides of various plants are presently known. However, only one polypeptide has been isolated from grapes and characterized. At the same time, tens of thousands of uncharacterized amino acid sequences have been revealed in this plant, among which there can also be precursors of oligopeptide regulators. Due to the scientific and practical importance of innate immunity of agricultural plants, we have undertaken structural and functional investigation of these sequences to identify new regulatory oligopeptides including antimicrobial agents. For this purpose, we elaborated a special method of computer analysis enabling comparison of primary structures of putative precursors of grape oligopeptides with amino acid sequences of known oligopeptides of other plants. Structural similarity served as the basis for prediction of potential functional properties. As a result, over 20 new structures of antimicrobial and other grape oligopeptides have been found.     

Spontaneous Onset of Homochirality in Oligopeptide Chains Generated in the Polymerization of N-Carboxyanhydride Amino Acids in Water

This article is concerned with the spontaneous onset of homochiral oligopeptide sequences. We will show that the polymerization of hydrophobic NCA (N-carboxyanhydride = cyclic anhydride)-amino acid racemates (i.e. tryptophane, leucine and isoleucine) in aqueous solution yields oligopeptides that are characterized by a high degree of homochiral sequences. Furthermore we will show that quartz enhances efficiently the mole fraction of oligopeptides with homochiral sequence by selectively adsorbing the more stereoregular oligopeptides from an aqueous solution of oligo-D,L-leucine. We find in particular that the mole fraction of the adsorbed homochiral 7mers is 17 times larger than the mole fraction calculated for a theoretical, random process. Experimentally the stereoisomer distribution for each oligomer length can be determined by the use of enantio-labeling and LC-MS (Liquid Chromatography-Mass Spectrometry). Furthermore, if we start the polymerization with an enantiomeric excess (e.e.) of 20% of L-leucine (L-amino acid: D-amino acid = 6:4, molar ratio) we observe a chiral amplification in the enantiomeric homochiral oligopeptides. We think that such processes are relevant to the chemical evolution of single handedness.     

Protein sequence modules

Conserved protein sequence segments are commonly believed to correspond to functional sites in the protein sequence. A novel approach is proposed to profile the changing degree of conservation along the protein sequence, by evaluating the occurrence frequencies of all short oligopeptides of the given sequence in a large proteome database. Thus, a protein sequence conservation profile can be plotted for every protein. The profile indicates where along the sequences the potential functional (conserved) sites are located. The corresponding oligopeptides belonging to the sites are very frequent across many prokaryotic species. Analysis of a representative set of such profiles reveals a common feature of all examined proteins: they consist of sequence modules represented by the peaks of conservation. Typical size of the modules (peak-to-peak distance) is 25-30 amino acid residues.     

Structural role of the polyglutamate portion of the folate found in T4D bacteriophage baseplate.

Three types of reagents were used to determine the structural role and location of the polyglutamate portion of the Escherichia coli T4D bacteriophage baseplate dihydropteroyl hexaglutamate. These reagents were examined for their effect in vitro on some of the final steps in phage baseplate morphogenesis. The reagents were (i) a series of oligopeptides composed solely of glutamic acid residues but with various chemical linkages and chain lengths; (ii) a homogeneous preparation of carboxypeptidase G1, an exopeptidase that hydrolyzes carboxyl-terminal glutamates (or aspartates) from simple oligopeptides, including the gamma-glutamyl bonds on folyl polyglutamates as well as the bond between the carboxyl group of the p-aminobenzoyl moiety and the amino group of the first glutamic acid residue of folic acid; and (iii) antisera prepared against a polyglutamate hapten. All three types of reagent markedly inhibited the attachment of the phage long tail fibers to the baseplate. Other steps in baseplate assembly such as the addition of T4D gene 11 or gene 12 products were not affected by any of these reagents. These results indicate that the polyglutamate portion of the folate is located near the attachment site on the bacteriophage baseplate for the long tail fibers.     

Protein Sequence Modules

Abstract

Conserved protein sequence segments are commonly believed to correspond to functional sites in the protein sequence. A novel approach is proposed to profile the changing degree of conservation along the protein sequence, by evaluating the occurrence frequencies of all short oligopeptides of the given sequence in a large proteome database. Thus, a protein sequence conservation profile can be plotted for every protein. The profile indicates where along the sequences the potential functional (conserved) sites are located. The corresponding oligopeptides belonging to the sites are very frequent across many prokaryotic species. Analysis of a representative set of such profiles reveals a common feature of all examined proteins: they consist of sequence modules represented by the peaks of conservation. Typical size of the modules (peak-to-peak distance) is 25–30 amino acid residues.     

The cardinal principle of like attracting like generates many ubiquitous oligopeptides shared by divergent proteins

Actual protein amino acid sequences are very different from random assemblages of 20 varieties of amino acids. The separate survey of 20 unrelated proteins in two steps that included eight of the 18 discussed in this paper, revealed that at the level of 5000 total residues, one out of every 32 tetrapeptides appeared in two or more identical copies, whereas at the level of 10 000 total residues, the frequency was elevated to one out of every 29. It would thus appear that only 60 000 or so, out of the possible 160 000 (20⁴) varieties of tetrapeptides, are regularly used by all proteins. These shall be defined as ubiquitous tetrapeptides. Those tetrapeptides occasionally found to be stray which did not belong to the above group of 60 000 must have been generated by new mutations. Thus, they are expected to return to the group by subsequent mutations. The above ubiquity is due to the cardinal principle of protein construction which is like attracting like. On the average, 28% of each residue is devoted to the formation of homodipeptides such as Leu-Leu, Asn-Asn and Trp-Trp. Consequently, homo-oligopeptides, pentapeptidic and longer, are readily found in two or more proteins unrelated to each other. The next in line among the ubiquitous oligopeptides are those made of similar residues. They usually contain palindromic cores such as Leu-Val-Leu, Ala-Gly-Ala and Lys-Arg-Lys. For example, the hexapeptide Ala-Gly-Ala-Asp-Ala-Ala is shared between human phosphofructokinase and bacterial cytochrome C. Provided that they are longer than 60 residues, all proteins contain repeating oligopeptides, tetrapeptidic to heptapeptidic in length. The above principle of like attracting like is the very reason that hydropathic profiles of most proteins readily yield alternating stretches of hydrophilic and hydrophobic segments.     

Biochemical problems of regulation by oligopeptides

Some problems are considered which arise in biochemical studies on structure and function of natural oligopeptides consisting of 2–50 amino acid residues. The problems under consideration include the generation of oligopeptides from precursors, chemical structure, the role of functionally important radicals and spatial configuration, and structure-function relationships. Different types of regulation are shown mainly for oligopeptides involved in muscle contraction.Translated from Biokhimiya, Vol. 69, No. 11, 2004, pp. 1565–1573.Original Russian Text Copyright © 2004 by Zamyatnin.     

Mass spectrometry of acetylated,permethylated and reduced oligopeptides

The amino acid sequence of oligopeptides can be determined from the mass spectra of their volatile derivatives. New peptide derivatives are described which are prepared from permethylated peptides by reduction with borane-tetrahydrofuran. These derivatives have been found to be more volatile than the corresponding permethylated ones. The procedure is illustrated by application of this technique to representative oligopeptides. As little as 10–100 nmol of these peptides have been sequenced using this technique.     

Hemoglobin as a potential source of natural regulatory oligopeptides

Theoretical structure-function analysis of all possible hemoglobin molecule fragments was performed to determine sites that could be potential sources of regulatory oligopeptides. Known data on bovine hemoglobin primary structure and information of the EROP-Moscow database concerning structure and functions of natural oligopeptides were used along with a computer program complex. A total of 6750 natural non-hemoglobin oligopeptides with hemoglobin fragments of 2–14 amino acid residues were found. Structures of 20 of them were completely identical to hemoglobin fragments. Most of the revealed oligopeptides exhibit properties of neuropeptides, antimicrobial agents, and hormones. A number of them exhibit functions previously not known for hemoglobin fragments. The possibility of natural formation of regulatory oligopeptides from hemoglobin and other food protein molecules, generation of the exogenous oligopeptide pool, their participation in regulation processes as well as accordance of results obtained here with the oligopeptide continuum concepts are discussed.     

Oligopeptides as plant growth regulators

It is generally accepted that plant growth and development are regulated by the known plant hormones. Some objections to the functions of auxins and cytokinins in the induction of shoot and root primordia are reported. Instead of them oligopeptides of special amino acid sequences could be the endogenous signals. There exist structure relationships between auxins and parts of the α-helical oligopeptides of defined amino acid sequences. The same is true for cytokinins. The most difficult part of this hypothesis is its verification. Using protonemata ofFunaria hygrometrica bud induction by various oligopeptides was investigated. The most active peptide tested is leucine-tryptophan. On the other hand endogenous oligopeptides containing [¹⁴C]-leucine in the moss protonemata during endogenous bud initiation were looked for. Three to four different oligopeptide spots seem to be related to bud induction.     

MUCOPOLYSACCHARIDES PRODUCED BY A STRAIN OF CLOSTRIDIUM PERFRINGENS.

Izumi, Kunihiko (Kanazawa University, Kanazawa, Japan). Mucopolysaccharides produced by a strain of Clostridium perfringens. J. Bacteriol. 83:956-959. 1962.-A new series of mucopolysaccharides was isolated from the culture medium of Clostridium perfringens and partially purified by the use of a column of anion-exchange resin. A large part of the substance was composed of neutral sugars, amino sugars, uronic acids, and oligopeptides, suggesting a structure analogous to that of bacterial cell walls. Acidic amino acids, especially aspartic acid, were the main constituents of the oligopeptides. The substance exhibited high viscosity when dissolved in water. The degree of viscosity in each fraction seemed to depend on the content of amino sugars and the chain length of the oligopeptides.     

Search for functional domain boundaries and localization of functional sites based on oligopeptide vocabularies]

A new method based on the analysis of oligopeptide composition of the amino acid sequences from different protein families is presented. We assume, that any protein family can be characterized by the set of oligopeptides (oligopeptides vocabulary). We demonstrate, that oligopeptides vocabulary comparison can distinguish different families from each other and from random sequences. It should be noted, that this comparison can be successfully performed on the set of only 25 dipeptides and without preliminary alignment. We demonstrate, that characteristic peptides are localized in the regions of functional significance, as shown on the example of GTP-binding domain of translation elongation factors. We suggest how to use this method to localize the boundaries of functional domains in amino sequences. On the example of few functional domains we demonstrate, that the average error of prediction does not exceed 3-4 amino acid residue.     

Characterization of new antihypertensive angiotensin I-converting enzyme inhibitory peptides from Korean traditional rice wine

This study describes the characterization of a new angiotensin I-converting enzyme (ACE) inhibitory peptide from a Korean traditional rice wine. After purification of the ACE inhibitor peptides with ultrafiltration, Sephadex G-25 column chromatography, and successively C?? and SCX solid-phase extraction, reverse-phase HPLC, and size exculsion chromatography, two types of the purified ACE inhibitors with IC?? values of 0.34 mg/ml and 1.23 mg/ml were finally obtained. The two purified ACE inhibitors (F-1 and F-2) were found to have two kinds of novel oligopeptides, showing very little similarity to other ACE inhibitory peptide sequences. The amino acid sequences of the two purified oligopeptides were found to be Gln- Phe-Tyr-Ala-Val (F-1) and Ala-Gly-Pro-Val-Leu-Leu (F-2), and their molecular masses were estimated to be 468.7 Da (F-1) and 357.7 Da (F-2), respectively. They all showed a clear antihypertensive effect on spontaneously hypertensive rats at a dosage of 500 mg/kg.