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The interaction of short nucleotide duplexes with bis-netropsins, in which netropsin fragments are linked in the tail-to-tail orientation via cis-diammineplatinum group (--Nt-Pt(NH3)-Nt--) or aliphatic pentamethylene chain (--Nt-(CH2)5-Nt--), has been studied. Both the bis-netropsins have been shown to bind to DNA oligomer 5'-CCTATATCC-3' (I) as a hairpin with parallel orientation of netropsin fragments in 1:1 stoichiometry. Monodentate binding has been detected upon binding of bis-netropsins to other duplexes of sequences 5'-CCXCC-3'--where X = TTATT (II), TTAAT (III), TTTTT (IV), and AATTT (V)--along with the binding of bis-netropsins as a hairpin. The formation of dimeric antiparallel motif between the halves of two bound bis-netropsin molecules has been observed in the complexes of--Nt-(CH2)5-Nt--with DNA oligomers IV and V. The ratio of binding constant of bis-netropsin as a hairpin (K2) to monodentate binding constant (K1) has been shown to correlate with the width and/or conformational lability of DNA in the binding site. The share of bis-netropsin bound as a hairpin decreases in the order: TATATTTATTTTAATTTTTTAATTT, whereas the contribution of monodentate binding rises. The minimal strong binding site for--Nt-Pt(NH3)-Nt--and--Nt-(CH2)5-Nt--binding as a hairpin has been found to be DNA duplex 5'-CGTATACG-3'.

De novo design and synthesis by a solid phase technique of linear and cyclic 26-residues peptides are reported. The peptides use beta-strand-turn-beta -strand motif for sequence recognition on DNA. Amino acid sequences in the two peptides are identical, but the structure of the cyclic peptide is constrained by S-S bridge between two cysteine residues. A 28-residue peptide containing at the N-terminus a copper-chelating peptide Gly-Gly-His is also synthesized which can be used as a potential DNA-cleaving reagent. Binding of these peptides to various natural and synthetic DNAs and DNA fragment with a known base pair sequence has been studied by CD spectroscopy, fluorescence methods and DNAse I footprinting technique. By means of CD spectroscopy it is shown that 26-residue linear and cyclic peptides are partially in disordered and beta-conformations in aqueous solution in absence and in presence of 20% trifluoroethanol (TFE), but assume partially an alpha-helix conformation in the presence of 50% TFE. It is shown that linear and cyclic peptides bind to DNA. The binding approaches saturation level when one peptide molecule is bound approximately per three or four DNA base pairs. We found that antibiotic distamycin A, binding in the minor DNA groove, competes effectively with the 26-residue linear and cyclic peptides for binding to poly(dA).poly (dT). According to the CD spectroscopy data the linear and cyclic peptides undergo conformation changes upon binding to DNA, whereas the DNA structure is not markedly altered. Difference CD spectra obtained by subtracting the spectrum of the free DNA from the spectrum of the peptide-DNA mixture differ from the spectrum of the free peptide. The shapes of difference CD spectra are consistent with a conformation transition from a disordered conformation into a beta-like conformation upon binding of peptide to DNA. DNAase I footprinting diagrams show that there is a specific protection by linear and cyclic peptides of the nucleotide sequences on two ends of operators OR1, OR2 and OR3 and pseudooperators within the cro gene of 434 phage.

Synthesis and DNA-binding activities of the two synthetic 26-residue peptides, containing in two copies a part of the DNA-binding region of the transcription activator v-Jun, are reported. Aminoacid sequences of the two peptides are identical, but in one of them the structure of the DNA-binding region is stabilized by S-S-bond between the two cysteine residues. Using CD spectroscopy, it is shown that the two peptides exist in a random coil conformation in aqueous solution, but assume partially an alpha-helical conformation in the presence of 20% trifluoroethanol. The percentage of alpha-helix is increased in the presence of 40% trifluoroethanol up to approximately 65% and 40% in the absence and presence of S-S-bond between the two cysteine residues, respectively. Evidently, formation of S-S-bond prevents a coil to alpha-helix transition in one of the two DNA-binding regions of the peptide, whereas the formation of alpha-helix in another DNA-binding region is allowed. It is shown that the two peptides bind to DNA. We found that the DNA minor groove-binding antibiotic distamycin A competes with the two peptides for binding to poly(dA).poly(dT). The binding of the two peptides to DNA is accompanied by conformational transitions in the peptide molecules, whereas the structure of DNA does not undergo a marked change. The difference CD spectrum obtained by subtracting the spectrum of DNA from the spectrum of a peptide-DNA mixture differs from the CD spectrum of the free peptide. The shapes of the difference CD spectra are consistent with alpha-beta and coil-beta transitions induced upon binding of the two peptides to DNA. DNase I footprinting diagrams show that peptides mediated cleavage protection of DNA takes place at regions containing 5'-TGA-3' and 5'-TGC-3' nucleotide sequences.

Synthesis and DNA-binding activity of the synthetic 26-residue peptide, containing in two copies a part of the DNA-binding domain of the transcription activator v-Jun, are reported. Using CD spectroscopy, it has been shown that the peptide exists in a random coil conformation in aqueous solution, but assumes partially an alpha-helical conformation in the presence of 20% trifluoroethanol. The percentage of alpha-helix is increased in the presence of 40% trifluoroethanol up to approximately 80%. It has been shown that the peptide forms two types of complexes with DNA. The first type of complexes saturates when one peptide molecule occupies six base pairs. At further increase of molar peptide to DNA ratio the binding became a cooperative process. The binding approaches saturation when one peptide molecule is bound approximately to four DNA base pairs. The binding constant of the monomer peptide complex with DNA has been estimated to be approximately 1.10(5) M-1 in the presence of 0.2 M NaCl. The peptide binds more strongly to poly(dG).poly(dC) and poly(dA).poly(dT) than to poly[d(GC)].poly[d(GC)]. We found that the DNA minor groove-binding antibiotic distamycin A competes effectively with the peptide for binding to poly(dA).poly(dT).

Synthesis, DNA- and zinc ion-binding activities of the synthetic 23-residue peptide, forming a part of the DNA-binding domain of yeast transcription activator GAL-4, are reported. In presence of zinc ions considerable changes in the shapes of the fluorescence and CD spectra of the peptide are observed. It is shown that the peptide forms complexes with zinc ions containing one metal ion per peptide molecule with association constants on the order of (1-2) x 10(6) M-1. Using gel filtration on a TSK-gel column we have shown that in aqueous solution at concentrations of 10(-4)-10(-6) M the peptide exists predominantly in the dimeric form. Dimerization constants were found to be 5 x 10(6) M-1 and 1.7 x 10(7) M-1 in the absence and in the presence of zinc ions, respectively. It is shown that the peptide binds to DNA. The binding approaches saturation when one peptide molecule is bound approximately to five base pairs of DNA. The shapes of the titration curves obtained from binding of the peptide to DNA show that the peptide can bind to DNA both in the monomeric and self-associated forms (dimer or tetramer). Increasing DNA concentration and decreasing the peptide/DNA molar ratio lead to a shift in the equilibria between self-associated peptide species and monomers toward the formation of monomer peptide complexes.

An analogue of netropsin has been synthesized consisting of two N-propylpyrrolcarboxamide units linked covalently to a copper-chelating tripeptide Gly-Gly-L-His by means of two and three glycine residues. Binding to DNA and synthetic polynucleotides of netropsin analogue containing three glycine residues between Gly-Gly-L-His tripeptide and the N-end of netropsin analogue (His-Nt) has been studied. It is shown that this netropsin analogue chelates a copper ion with 1:1 stoichiometry, similar to a free Gly-Gly-L-His peptide. It is found that this netropsin analogue occupies 3 to 4 base pairs upon binding to poly(dA).poly(dT) and poly[d(AT)].poly[d(AT)] polymers, irrespective of whether it binds in Cu(2+)-ligated or unligated forms. Binding constants and binding site sizes have been calculated for netropsin analogue complexes with DNA, poly(dA).poly(dT) and poly[d(AT)].poly[d(AT)] polymers at the [Cu2+]/[His-Nt] ratio equal to 0 and 1.0. In the three-component system including His-Nt and Cu(2+)-His-Nt, cooperative effects are recognized which can be explained by heterodimer generation on interaction of His-Nt and Cu(2+)-His-Nt at adjacent binding sites.

Experimental data are reported on DNA-cleaving activity of the synthetic netropsin analogs consisting of the two N-propylpyrrole carboxamide units linked covalently through two or three glycine residues to a copper-chelating tripeptide glycyl-glycyl-L-histidine. Incubation of DNA restriction fragment and netropsin analog in the presence of ascorbate, hydrogen peroxide and Cu2+ ions resulted in selective cleavage of the DNA at or near the preferred sites for binding of netropsin analog. A similar cleavage pattern is observed after X-ray irradiation of DNA complexes with netropsin analogs tethered with Cu2+ ions. The cleavage patterns are found to be dependent on the length of the connecting chain between the histidine-containing tripeptide and netropsin analog. The netropsin analog containing three glycine residues in the connecting chain, but not the analog with a shorter linker chain, can generate an intense cleavage of one of the two polynucleotide chains at a position corresponding to the presumed binding site for the dimeric ligand species. More than 50% of the total DNA can be cleaved at this position after X-ray irradiation. From analysis of the nucleotide sequences surrounding the preferred cleavage site on several DNA fragments we found that the consensus is 5'-TTTTNCA*AAA-3', where N is an arbitrary nucleotide. The Cu(2+)-mediated cleavage of DNA occurs at the second adenine (indicated by an asterisk) from the 5'-end of the sequence. The greatest cleavage activity is observed when the molar ratio of Cu2+ to the netropsin analog is equal to 0.5. Evidently, the Cu(2+)-ligated and unligated oligopeptide species interacts with each other to form a heterodimer bound to DNA at the cleavage site. To test the validity of this model we have studied the binding of unligated netropsin analog and netropsin analog complexed with Cu2+ ion to a self-complementary oligonucleotide 5'-GCGTTTTGCAAAACGC-3'. It is found that binding of Cu(2+)-ligated netropsin analog to the DNA oligomer preincubated with unligated form of the oligopeptide is a cooperative process for which interactions between the two bound ligands are responsible. The cooperativity parameter is estimated to be on the order of factor 6. Finally, a model is proposed in which a heterodimer stabilized by interligand beta-sheet binds in the minor DNA groove.

Cystine peptide dimer (Lys-Gly-Val-Cys-Val-N2H2Dns)2 with S-S bridge was synthesized and its interactions with DNA and synthetic polynucleotides have been studied by optical spectroscopy methods. By recording fluorescent titration curves we have shown that the affinity of the peptide to different synthetic polynucleotides decreases in the order: poly(dG).poly(dC) greater than poly(dA).poly(dT) greater than poly(dGC).poly(dGC). The stability of complexes to increasing concentrations of NaCl diminishes in the same order. The association constant is about 20-fold greater for peptide binding to poly(dG).poly(dC) than to poly(dA).poly(dT). By using circular dichroism and fluorescence measurements we have shown that the peptide competes for the binding sites on DNA with two minor-groove binding antibiotics--distamycin A and sybiromycin. These results have suggested that the peptide also binds in the DNA minor groove. Investigation of the interactions between such peptides and DNA may be useful for constructing ligands with combined specificity to DNA.

Binding of synthetic pentapeptide Val-Thr-Thr-Val-Val-N2H2Dns (where Dns is a residue of 5-dimethylamino naphthyl-1-sulfonic acid) is studied by circular dichroism, electron microscopy and fluorescence methods. It is found that this peptide can self-associate in aqueous solution as revealed from the concentration-dependent changes in the UV absorbance and fluorescence spectra. At high peptide concentration (3.10(-4) M) massive peptide aggregates are formed in solution and can be visualized by electron microscopy. It is shown that pentapeptide binds to DNA predominantly in a self-associated form and exhibits preferences for certain nucleotide sequences. It binds more strongly to poly(dG).poly(dC) and poly[d(A-C)].poly[d(G-T)] than to poly(dA).poly(dT). The complex with poly(dA).poly(dT) dissociates in the presence of 0.05 M NaCl, whereas the complex with poly(dG).poly(dC) is stable even in the presence of 0.2 M NaCl. The binding is a cooperative process which is accompanied by compaction of DNA at peptide/DNA base pair ratios greater than 2. At the initial stage of the compaction process the coalescence of DNA segments covered by bound peptide molecules results in the formation of DNA loops stabilized by interaction between bound peptide molecules. Increasing peptide/DNA ratio leads to the formation of rod-like particles as revealed from electron microscopy studies. Further increase in the peptide concentration leads to folding of fibrillar macromolecular complexes into globula each containing a single DNA molecule.

Design, synthesis and DNA binding activity of a nonlinear 102 residue peptide are reported. The peptide contains four sequence-specific DNA binding domains of 434 Cro protein. These four domains were linked covalently to a symmetrical carboxyterminal crosslinker that contains four arms each ending with an aliphatic aminogroup. From CD studies we have found that in aqueous buffer in the presence of 20% trifluoroethanol the peptide residues assume alpha helical, beta-sheet and random coiled conformations with an alpha helical content of about 16% at room temperature. The alpha helicity is increased up to 40% in the presence of 40% trifluoroethanol. Upon complex formation between the peptide and DNA a change in the peptide conformation takes place which is consistent with an alpha-beta transition in the DNA binding, helix-turn-helix motif of 434 Cro repressor. Evidently residues present in helices alpha(2) and alpha(3) form a beta hairpin which is inserted in the minor DNA groove. The latter inference is supported by our observations that the peptide can displace minor groove binding antibiotic distamycin A from a complex with poly(dA).poly(dT). As revealed from DNase protection studies the peptide exhibits preferences for binding to operator and pseudooperator sites recognized by 434 Cro repressor. It binds strongly to operator sites OR1, OR2 and OR3 and exhibits a greater affinity for pseudooperator site Op1. From analysis of nucleotide sequences in the strong affinity binding sites for the peptide on DNA a conclusion is drawn that it binds to pseudosymmetrical nucleotide sequences 5'-ACAA(W)nCTGT-3', where W is an arbitrary nucleotide. n is equal to six or seven. In the strongest affinity binding site for the peptide on DNA (Op1) motif 5'-ACAA-3' is replaced by sequence 5'-ACCA-3'. A difference in binding specificity shown by the peptide and 434 Cro protein could be attributed to a flexibility of the connecting chains between DNA-binding domains in the peptide molecule as well as to a replacement of Thr - Ala in the alpha 2 helix. Removal of two residues from the N-terminal end of helix alpha 2 in each of the four DNA binding domains of 434 Cro present in the peptide leads to a loss of binding specificity, although the modified peptide binds to DNA unspecifically.

The DNA binding activity of trivaline dansyl hydrazide was investigated by circular dichroism, UV spectrophotometry and fluorescence methods. It is shown that these peptides in the absence of DNA can adopt statistical coil and antiparallel beta-conformation and can exist in aqueous solution as monomers, dimers and higher orders aggregates depending upon the concentration and the presence of N- and C-blocking groups. The aggregation and disaggregation processes are very slow especially for N- and C-protected peptides. Our observations show that oligopeptides in the monomeric and dimeric forms bind to double-stranded DNA and RNA whereas tetramers and higher order aggregates exhibit no DNA binding activity. The binding of monomers is a cooperative process favoring the formation of deformed antiparallel beta-structure between adjacently bound monomers. The binding constant of dimeric oligovaline species to GC-rich DNA sequences is about 5 fold higher than that found from the binding of oligovaline to poly(dA) . poly(dT). The binding of dimers takes place in the minor DNA groove as revealed from our observations that oligovaline binds to T6 phage DNA containing massive glucose and diglucose residues in the major groove. The backbone C=0 groups of oligovaline probably serve as specific reaction centres for the interaction with guanine 2-amino groups in the minor DNA groove.

In the present communication design, synthesis and DNA binding activities of three bis-netropsins and two netropsin analogs containing two N-propylpyrrolecarboxamide fragments linked covalently to peptides Gly-Gly-(analog I) and Val-Val-Val-Gly-Gly-(analog II) are reported. Each bis-netropsin consists of two netropsin-like fragments attached to peptides -Gly-Cys-Gly-NH2 (compound IIIa), H-Gly-Cys-Gly-Gly-Gly-(compound IV) or Gly-Cys-Sar-NH2 (compound IIIb) which are linked symmetrically via S-S bonds. Physico-chemical studies show that each bis-netropsin carries 6 AT-specific reaction centers and covers approximately 10 base pairs upon binding to poly(dA).poly(dT). This indicates that two netropsin-like fragments of the bis-netropsin molecule are implicated in specific interaction with DNA base pairs. The peptide fragments of bis-netropsins IIIa and IV form small beta-sheets containing two-GC-specific reaction centers. The DNase I cleavage patterns of bis-netropsin-DNA complexes visualized by high resolution gel electrophoresis show that the preferred binding sites for bis-netropsins IIIa and IV are identical and contain two runs of three or more AT pairs separated by two GC pairs. Specificity determinants of netropsin analog II binding in the beta-associated dimeric form are identical to those of bis-netropsin IIIa thereby indicating that there is a similarity in the structure of complexes formed by these ligands with DNA. In the monomeric form analog II exhibits binding specificity identical to that of analog I. Replacement of C-terminal glycine residues by sarcosines in the peptide fragments of bis-netropsin IIIa leads to a decrease in the affinity of ligand for DNA.