Recognition of damaged regions in DNA by oligopeptides and proteins

The binding of various damaged DNAs to the single-strand binding protein coded for by gene 32 from bacteriophage T4, on the one hand, and of oligopeptides containing tryptophan and lysine residues, on the other hand, is described. These molecules exhibit a higher affinity for modified DNA than for native DNA in so far as modification results in a local destabilization of the double-stranded structure of the nucleic acid. Stacking interactions between aromatic amino acids and nucleic acid bases appear to play a crucial role in the recognition of destabilized regions induced by chemical agents (carcinogens and antitumor drugs). These interactions confer to the peptide lysyl-tryptophyl-lysine an endonucleolytic activity specific for apurinic sites. From results obtained with such oligopeptides a model for the active sites of Ap-endonucleases is proposed which could account for the strategy used by the denV endonuclease from phage T4 during the first step of excision repair of pyrimidine dimers in DNA. The effect of the overall conformation of modified DNA on repair efficiency is discussed.     

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.     

Multiple peptide synthetase gene clusters in Actinomycetes

Two oligonucleotide probes derived from conserved motifs in peptide synthetases were hybridized with a cosmid library of Planobispora rosea genomic DNA. Detailed characterization of the physical organization of the positive cosmids indicated the existence of at least eight unlinked contigs containing multiple fragments that hybridized to both probes. Partial sequences of PCR products from the positive cosmids confirmed the existence of peptide synthetase genes. The combined results of hybridizations and physical mapping indicate that, in all likelihood, the isolated P. rosea contigs encode over 40 putative peptide synthetase modules. Similar results were obtained on screening a cosmid library of Actinoplanes teichomyceticus DNA. Furthermore, Southern hybridizations with several actinomycete strains, belonging to different genera, indicate that most strains contain multiple hybridizing bands well in excess of the number expected from the structure of the oligopeptides produced by these strains. Even strains not reported to produce oligopeptides gave clear positive signals when examined with the probes. These results strongly suggest that actinomycetes devote a notable fraction of their genomes to the non-ribosomal synthesis of peptides, and that most strains have the genetic potential to produce more oligopeptides than are currently described.     

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.     

Nucleoprotein fusion. III. Causes and conditions for development of the multiphase character of the DNA--protein fusion curve

The influence of proteins reversibly and irreversibly bound to DNA on the shape of melting curve has been considered. It is shown that the melting curve becomes biphasic in two cases: (i) cooperative binding of proteins with DNA (II) STRONG DIFFERENCE IN THE BINDING CONSTANTS WITH HELICAL AND COILED REGIONS. Simple formulae permitting to determine which of two causes stipulate for biphasic profile of a given experimental melting curve are obtained. Melting curves of DNA-basic oligopeptides complexes have been investigated. It is shown that the oligopeptides, when their chain length does not exceed 10, are able to migrate along DNA and biphasic shape of the melting curve is stipulated by the cooperative manner of their binding with DNA.     

Self-organization of oligopeptides obtained on dissolution of feather keratins in superheated water

Keratins are self-organized proteins that are abundantly available in wool, feather, human hair, etc., making them a potential cheap feedstock for the modification of amino acids. This paper explores the hydrolysis of keratin in water under specific pressure-temperature conditions where the hydrolysis through scission of the protein chain yields oligopeptides. Here we report for the first time that, under appropriate conditions, these oligopeptides self-assemble into a hierarchical architecture, the process being followed in time by optical microscopy. Birefringent needle-like crystals are observed which tend to nucleate heterogeneously. When given sufficient time, these needles become tens of microns in length and act as further nuclei, developing a highly repetitive structure of several hundreds of microns in size. Micro-focus X-ray diffraction studies supported by in situ microscopy reveal that these needles have a crystal structure similar to that of the native protein, although better organized along the ab-plane. Spectroscopic studies on these structures show crystalline bands that disappear above 150 degrees C, coinciding with an endothermic peak in DSC. Amino acid analysis shows that the self-assembled birefringent entities are indeed oligopeptides, consisting of sequences of approximately 40 amino acids. The proposed ecofriendly route provides an effective route for obtaining oligopeptides that can be used as important building blocks for the synthesis of a range of novel polymers. The oligopeptides obtained from the sustainable source can be used as important building blocks for the synthesis of a range of novel polymers.     

DNA packaging via combinative self-assembly

A novel and versatile DNA packaging approach was developed by grafting DNA-binding oligopeptides onto a polymer scaffold to combinatively self-assemble with DNA into compact nanostructures.     

H4 histone tail mediated DNA-DNA interaction and effects on DNA structure, flexibility, and counterion binding: a molecular dynamics study

All-atom molecular dynamics (MD) simulations were performed during 30-45 ns for a system of three identical DNA 22-mers, 14 short fragments of the charged H4 histone tail peptide fragment (amino acids 5-12, KGGKGLGK) with K(+) counterions, and explicit water. The simulation setup mimics the crowded conditions of DNA in eukaryotic chromatin. To assess the influence of tail fragments on DNA structure and dynamics, a "control" 20 ns MD simulation was carried for a system with the same DNA and water content but in the absence of oligopeptides. Results of DNA interaction with the histone tail fragments, K(+), and water is presented. DNA structure and dynamics and its interplay with the histone tail fragments binding are described. The charged side chains of the lysines play a major role in mediating DNA-DNA attraction by forming bridges and coordinating to phosphate groups and electronegative sites in the minor groove. Binding of all species to DNA is dynamic. Some of the tail fragments while being flexible and mobile in each of its functional groups remain associated near certain locations of the DNA oligomer. The present work allows capturing typical features of the histone tail-counterion-DNA structure, interaction, and dynamics.     

Inhibition of human DNA topoisomerase I by new DNA minor groove ligands: derivatives of oligo-1,3-thiazolecarboxamides.

A series of novel thiazole-containing oligopeptides (oligo-1,3-thiazolecarboxamides) interesting specifically with the minor groove of DNA was shown to inhibit human DNA topoisomerase I (topo I). Inhibitory effects of thiazole-containing oligopeptides (TCO) increase with the number of thiazole units in such compounds. Inhibitory properties of TCO containing 3 or 4 thiazole units were shown to be 3-10 times better than those of the well-known natural antibiotic, distamycin A containing pyrrole rings. The structure of various additional groups attached to the N-terminus and C-terminus of TCO had no significant effect on TCO interaction with the complex of DNA and topo I. TCO were shown to be capable of binding with double-stranded DNA (dsDNA), and the majority of TCO analyzed were more effective in binding with dsDNA than distamycin A. Possible reasons for the different effects of distamycin A and TCO on the reaction of relaxation catalyzed by topo I are discussed.     

Oligopeptides in the development of biological motivations

Data are presented, demonstrating the action of a number of oligopeptides on biological motivations of hunger, fear, self-stimulation and on alcohol addiction. In the structure of animals feeding motivation, such oligopeptides take part as beta-lipotropin and its fragments, ACTH, pentagastrin, delta-sleep inducing peptide (DSIP), substance P; in organization of defensive motivation--angiotensin II (AII), DSIP, substance P, bradykinin, beta-endorphin etc.; in organization of self-stimulation--AII, DSIP, bradykinin, ACTH, beta-endorphin etc. It is established that most of the above oligopeptides, injected to the brain lateral ventriculi, inhibit biological motivations, and only some of them have an activating action. On the basis of experiments, a hypothesis is formulated that oligopeptides act as a feedback between the genome of brain neurones and pacemaker cells of motivation centres of the hypothalamus area. Some oligopeptides elaborated by neuronal genomes under the action of dominating motivation, activate--and the other--suppress the activity of motivation hypothalamus centres.     

Cationic oligopeptides modified with lipophilic fragments: use for DNA delivery to cells

Cationic oligopeptides, including the amphipathic alpha-helical peptides, are applied to the targeted delivery of DNA to eukaryotic cells due to their DNA-compacting properties and the ability to destabilize the cell lipid bilayer in some cases. We synthesized the peptides differing in the number and location of residues of decanoic acid covalently attached to Lys residues in order to combine the DNA-binding and the membrane activities in a single molecule. We chose peptide structures that assisted in the formation of alpha-helices. The DNA-binding ability of the peptides and the membrane activity of their complexes with DNA were shown to depend on the structure. The study of erythrocyte hemolysis by complexes with DNA of the pCMV LacZ plasmid and the peculiarities of transfection of these complexes revealed a correlation between the hemolytic activity and the expression level of the lacZ gene in the cells.     

DNA recognition by synthetic peptides as a model for the dimeric proteins.

A Chiral template possessing a C2 axis has been utilized to study an effect in the dimer formation of oligopeptides. Oligopeptide rich in basic amino acids are used as subunits that would interact with DNA, and the subunits are aligned according to the C2 axis of the template. Synthesis, characterization and DNA binding study of these dimeric peptides will be discussed.     

The place of carnosine among physiologically active peptides]

The results of analysis of EROP-Moscow data base concerning structural and functional peculiarities of endogenous regulatory oligopeptides are reviewed in relation to carnosine, the first endogenous peptide bioregulator. The dipeptide fragment ala-his is widely distributed in natural systems, in particular in various representatives of living organisms. The main structural peculiarity of carnosine is its elongated "filamentous" structure with a positively charged N-terminus and a cyclic radical characteristic of large physiologically active oligopeptides. The relatedness of carnosine to other oligopeptides also becomes apparent during the analysis of its role in various regulatory systems of the body.     

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.     

Aminopeptidase P from rat brain. Purification and action on bioactive peptides.

Aminopeptidase P (EC 3.4.11.9) was purified from rat brain cytosol. A subunit Mr of 71,000 was determined for the reduced, denaturated protein whereas an Mr of 143,000 was determined for the native enzyme. The purified aminopeptidase P selectively liberated all unblocked, preferentially basic or hydrophobic ultimate amino acids from di-, tri- and oligopeptides with N-terminal Xaa-Pro- sequences. Corresponding peptides with penultimate Ala instead of Pro were cleaved with much lower rates; oligopeptides with residues other than Pro or Ala in the penultimate position appeared not to be substrates for the enzyme. Several bioactive peptides with Xaa-Pro sequences, especially bradykinin, substance P, corticortropin-like intermediate lobe peptide, casomorphin and [Tyr]melanostatin were shortened by the N-terminal amino acid by aminopeptidase P action. Rat brain aminopeptidase P was optimally active at pH 7.6-8.0 in the presence of Mn2+. Chelating agents and SH-reacting reagents inhibited the enzyme, but common inhibitors of aminopeptidases, like amastatin or bestatin, of prolidase or of dipeptidyl peptidases II and IV, like N-benzoyloxycarbonyl-proline or epsilon-benzyl-oxycarbonyl-lysyl-proline, as well as antibiotics like beta-lactam ones, bacitracin or puromycin, had little or no effect.     

A stand-alone adenylation domain forms amide bonds in streptothricin biosynthesis

The streptothricin (ST) antibiotics, produced by Streptomyces bacteria, contain L-β-lysine ((3S)-3,6-diaminohexanoic acid) oligopeptides as pendant chains. Here we describe three unusual nonribosomal peptide synthetases (NRPSs) involved in ST biosynthesis: ORF 5 (a stand-alone adenylation (A) domain), ORF 18 (containing thiolation (T) and condensation (C) domains) and ORF 19 (a stand-alone A domain). We demonstrate that ST biosynthesis begins with adenylation of L-β-lysine by ORF 5, followed by transfer to the T domain of ORF 18. In contrast, L-β-lysine molecules adenylated by ORF 19 are used to elongate an L-β-lysine peptide chain on ORF 18, a reaction unexpectedly catalyzed by ORF 19 itself. Finally, the C domain of ORF 18 catalyzes the condensation of L-β-lysine oligopeptides covalently bound to ORF 18 with a freely diffusible intermediate to release the ST products. These results highlight an unusual activity for an A domain and unique mechanisms of crosstalk within NRPS machinery.     

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.