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A two-step synthesis for methionine-containing hydrophobic and/or aggregation-prone peptides is presented that takes advantage of the reversibility of methionine oxidation. The use of polar methionine sulfoxide as a building block in solid-phase peptide synthesis improves the synthesis quality and yields the crude peptide, with significantly improved solubility compared to the reduced species. This facilitates the otherwise often laborious peptide purification by high-performance liquid chromatography. The subsequent reduction proceeds quantitatively. This approach has been optimised with the methionine-rich Tar-DNA-binding protein 43 (307-347), but is also more generally applicable, as demonstrated by the syntheses of human calcitonin and two aggregation-prone peptides from the human prion protein., (© 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Disulfide bond-containing peptides are useful molecular scaffolds with diagnostic and therapeutic applications due to their good biological activity and good target selectivity, but their utility is sometimes limited by the lability of the disulfide moiety under reducing conditions and in the presence of disulfide bond isomerase. The development of disulfide surrogates with improved redox stability has been an area of ongoing research; and one possible strategy is based on a diaminodiacid (DADA) moiety, which can be used to synthesize the disulfide bond replacement peptides with precise structures and enhanced stability through automated solid-phase peptide synthesis (SPPS). This review summarizes recent developments in the DADA-based SPPS of peptide disulfide surrogates. Some representative applications and structural studies on the DADA-based disulfide surrogates are described., (© 2020 Wiley-VCH GmbH.)

One of the condensation methods for the preparation of long-chain peptides, the so-called thioester method requires protecting groups for amino and thiol groups for regioselective ligation. In this study, we demonstrated that the phenacyl (Pac) group acts as an efficient protecting group of cysteine side chains. We synthesized a cysteine derivative carrying the Pac group at the side chain sulfur atom, and Pac-containing peptides and peptide thioesters were synthesized using it by the ordinary 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase peptide synthesis strategy. Pac-containing peptide segments could be condensed by the thioester method. After the condensation reaction, Pac groups could be removed by Zn/AcOH treatment. In addition, the azido group, which was used for the protection of lysine side chains, was simultaneously converted into an amino group, demonstrating that this protecting group scheme simplified the deprotecting reaction after the peptide condensation reaction to a single step.

Synthetic light-driven molecular motors are molecular machines capable of rotation under photo-irradiation. In this paper, we report the synthesis of peptide-conjugated molecular motors and evaluate their DNA-binding properties.

In this study, two types of BolA-like amphiphilic peptides with dual ligands comprising a tumor-targeting moiety of RGD sequence and a cell-penetrating moiety of R8 sequence are designed and synthesized as gene vectors. The BolA-structural peptide carriers can self-assemble into spherical nanoparticles with a hydrophilic core and shell, which are similar to the viral capsid and can bind plasmid DNA in an aqueous medium to form viral-mimetic complexes. It is found that the BolA-like dual ligands system exhibits significantly enhanced gene expression in both HeLa and 293T cell lines, as compared with poly(ethylenimine) PEI. These BolA-like amphiphilic peptides are promising in clinical trials of gene therapy., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Although many zinc finger motifs have been developed to recognize specific DNA triplets, a rational way to selectively skip a particular non-recognized gap in the DNA sequence has never been established. We have now created a 6-zinc finger peptide with an alpha-helix linker, Sp1ZF6(EAAAR)4, which selectively binds to the discontinuous recognition sites in the same phase (10 bp gap) against the opposite phase (5 bp gap) of the DNA helix. The linker peptide (EAAAR)4 forms an alpha-helix structure stabilized by salt bridges, and the helical length is estimated to be about 30 A, corresponding to that of the 10 bp DNA. The gel shift assays demonstrate that Sp1ZF6(EAAAR)4 preferably binds to the 10 bp-gapped target rather than the 5 bp-gapped target. The CD spectra show that the alpha-helical content of the (EAAAR)4 linker is higher in the complex with the 10 bp-gapped target than in the complex with the 5 bp-gapped target. The present results indicate that the alpha-helical linker is suitable for binding to the recognition sites in the same phase and that the linker induces the loss of binding affinity to the recognition sites with the opposite phase. The engineering of a helix-structured linker in the 6-zinc finger peptides should be one of the most promising approaches for selectively targeting discontinuous recognition sites depending on their phase situations.

To identify rules for the design of efficient CPPs that can deliver therapeutic agents such as nucleic acids (DNAs, siRNAs) or proteins and PNAs into subcellular compartments, we compared the properties of several primary and secondary amphipathic CPPs. Studies performed with lipid monolayers at the air-water interface have enabled identification of the nature of the lipid-peptide interactions and characterization of the influence of phospholipids on the ability of these peptides to penetrate into lipidic media. Penetration and compression experiments reveal that both peptides interact strongly with phospholipids, and observations on Langmuir-Blodgett transfers indicate that they can modify the lipid organization. Conformational investigations indicate that the lipid-peptide interactions govern the conformational state(s) of the peptides. On the basis of the ability of both peptides to promote ion permeation through both natural and artificial membranes, models illustrating the translocation processes have been proposed. One is based on the formation of a beta-barrel pore-like structure while another is based on the association of helices.

Sequence-specific DNA binding of short peptide dimers derived from a plant basic leucine zipper protein EmBP1 was studied. A homodimer of the EmBP1 basic region peptide recognized a palindromic DNA sequence, and a heterodimer of EmBP1 and GCN4 basic region peptides targets a non-palindromic DNA sequence when a beta-cyclodextrin/adamantane complex is utilized as a dimerization domain. A homodimer of the EmBP1 basic region peptide binds the native EmBP1 binding 5'-GCCACGTGGC-3' and the native GCN4 binding 5'-ATGACGTCAT-3' sequences with almost equal affinity in the alpha-helical conformation, indicating that the basic region of EmBP1 by itself has a dual recognition codes for the DNA sequences. The GCN4 basic region peptide binds 5'-ATGAC-3' in the alpha-helical conformation, but it neither shows affinity nor helix formation with 5'-GCCAC-3'. Because native EmBP1 forms 100 times more stable complex with 5'-GCCACGTGGC-3' over 5'-ATGACGTCAT-3', our results suggest that the sequence-selectivity of native EmBP1 is dictated by the structure of leucine zipper dimerization domain including the hinge region spanning between the basic region and the leucine zipper.

Engineered zinc finger proteins revealed that a linker sequence connecting zinc finger units has a significant effect on the DNA binding property of the protein. The recognition for a noncontiguous DNA target beyond the current recognition code of zinc finger proteins has never been determined because of the limitation of a zinc finger framework. DNA recognition of zinc finger proteins is limited only to a contiguous subset of three base pairs. We propose the recognition for a noncontiguous DNA target by inserting amino acids into the canonical linker between zinc finger units. The sequence selectivity of the new zinc finger peptides was evaluated by gel mobility shift assays. DNase I footprinting analyses clearly showed different DNA binding of various linker-extended zinc finger peptides. The application of a SPR measurement also revealed a DNA sequence selectivity of peptides. Insertion of three amino acids is enough for recognition of a noncontiguous DNA target with sequence selectivity. An extended linker will be useful for expansion of the recognition code of zinc finger proteins and for development of a new role for linker sequences in DNA binding of zinc finger proteins.

Eukaryotic aminoacyl-tRNA synthetases have dispensable extensions appended at the amino- or carboxyl-terminus as compared to their bacterial counterparts. While a synthetic peptide corresponding to the basic amino-terminal extension in yeast Asp-tRNA synthetase binds to DNA, the extension in the intact protein evidently binds to tRNA and enhances the tRNA specificity of Asp-tRNA synthetase. On the other hand, the amino-terminal extension in human Asp-tRNA synthetase, both within the intact protein and as a synthetic peptide, binds to tRNA. Here, the tRNA binding of a synthetic peptide, hKRS(Arg(25)-Glu(42)), corresponding to the amino-terminal extension of human Lys-tRNA synthetase (hKRS) was analyzed. This basic peptide bound to tRNA(Phe) and the apparent-binding constant increased with increasing concentrations of Mg(2+). The hKRS peptide also bound to DNA and polyphosphate; however, the apparent DNA-binding constants decreased at increasing concentrations of Mg(2+). The ability of the hKRS peptide to adopt alpha-helical conformation was demonstrated by NMR and circular dichroism. A Lys-rich peptide derived from the elongation factor 1alpha was also examined and bound to DNA but not to tRNA.

We have designed new basic amphiphilic peptides, ppTG1 and ppTG20 (20 amino acids), and evaluated their efficiencies in vitro and in vivo as single-component gene transfer vectors. ppTG1 and ppTG20 bind to nucleic acids and destabilize liposomes consisting of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and cholesterol (3:1 mol/mol) at pH 5 and pH 7. Complexes of plasmid DNA and ppTG1 gave rise to efficient transfection in a variety of human and murine cell lines at low charge ratios ([+/-] between 1 and 2). In cell culture experiments, such vectors were superior to the membrane-destabilizing peptide KALA. In comparison with cationic lipid-, dendrimer-, and polymer-based transfection agents like Superfect, polyethylenimine (PEI), and Lipofectin, ppTG1 vectors showed good transfection efficiencies, especially at low DNA doses. Moreover, we demonstrated for the first time successful gene transfer in living animals with a single-component peptide vector. In the mouse, intravenous injection of a luciferase expression plasmid complexed with ppTG1 or ppTG20 led to significant gene expression in the lung 24 hours after injection. Structure-function studies with ppTG1, ppTG20, and sequence variants suggest that the high gene transfer activity of these peptides is correlated with their propensity to exist in alpha-helical conformation, which seems to be strongly influenced by the nature of the hydrophobic amino acids.

We have systematically designed and synthesized six kinds of 16-17 mer alanine-based peptides containing four to six lysine (K) and one to four asparagine (N) residues to achieve the selective binding to A.T base pairs of DNA duplexes. The position and number of K and N residues were changed in the helical structure according to common features of the DNA-binding proteins, in which K and N residues are expected to interact electrostatically with phosphate groups and to interact with A.T base pairs by hydrogen bonding, respectively. The time courses of binding of these peptides to dA(30).dT(30) and dG(30).dC(30) duplexes immobilized on a 27 MHz quartz crystal microbalance (QCM) were studied in 10 mM phosphate buffer (pH 7.5) and 40 mM NaCl at 10 degrees C. The maximum binding amounts (Deltam(max)) on a nanogram scale and binding constants (K(a)) could be obtained from the frequency decrease (mass increase) of the oligonucleotide-immobilized QCM. The conformation changes of the peptides upon binding to DNAs were monitored by circular dichroism (CD) spectroscopy. The four properly arranged N residues in the six-cationic K peptide, K6N4(d), resulted in a 5-fold higher affinity for A.T base pairs (K(a) = 5.9 x 10(5) M(-1)) than for G.C base pairs (K(a) = 1.2 x 10(5) M(-1)), and alpha-helices were clearly promoted by the binding to A.T base pairs from CD spectral changes.

The pAntp peptide, corresponding to the third helix of the homeodomain of the Antennapedia protein, enters by a receptor-independent process into eukaryotic cells. The interaction between the pAntp peptide and the phospholipid matrix of the plasma membrane seems to be the first step involved in the translocation mechanism. However, the mechanism by which the peptide translocates through the cell membrane is still not well established. We have investigated the translocation ability of pAntp through a protein-free phospholipid membrane in comparison with a more amphipathic analogue. We show by fluorescence spectroscopy, circular dichroism, NMR spectroscopy, and molecular modeling that pAntp is not sufficiently helically amphipathic to cross a phospholipid membrane of a model system. Due to its primary sequence related to its DNA binding ability in the Antennapedia homeodomain-DNA complex, the pAntp peptide does not belong to the amphipathic alpha-helical peptide family whose members are able to translocate by pore formation.

T22 ([Tyr5,12, Lys7]-polyphemusin II) is a strong anti-HIV compound. Six analogs of T22 and two natural forms were synthesized. Of them, all downsized peptides (14 residues; TW70, T131, T134, and T140) showed a higher selectivity index than did other, 17- or 18-residue peptides. In particular, T134 and T140 showed both lower cytotoxicity and higher antiviral activity than did T22 against HIV infection of MT-4 cells, an HTLV-I-bearing T cell line. To clarify the inhibitory mode of T22 and its analogs, we used a single-round replication assay (luciferase assay), in which different envelope-bearing pseudotypes were used to infect CXCR4- or CCR5-bearing U87 cells via CD4. All of the analogs inhibited T cell line-tropic strain HXB-2 (X4) and dual-tropic strain 89.6 (R5X4) HIV infections mediated by CXCR4, but had no effect on macrophage-tropic strain ADA (R5) or 89.6 HIV infections mediated by CCR5. The inhibition by T134 (IC50 of 2.70 nM) and T140 (IC50 of 0.432 nM) was also stronger than that by T22 (IC50 of 5.05 nM). The binding of anti-CXCR4 monoclonal antibody 12G5 to lymphoma-derived T cell line Sup-T1 was more efficiently blocked by T134 and T140 than by T22. Taken together, T22 and its analogs T134 and T140 exerted their inhibition by specific binding to CXCR4. The marked increase in the anti-HIV activity of T134 and T140 was ascribed to an enhancement in their ability to bind to CXCR4.

Sequence-specific DNA binding proteins generally consist of more than two DNA-contacting regions to ensure the selectivity of recognition. The multiple DNA binding modules are connected either through the covalent linker or through the noncovalent dimerization domain. We have compared the DNA binding of peptide dimers with covalent and noncovalent dimerization domains to explore the potential advantage of each linkage on the sequence-specific DNA binding. Three sets of head-to-tail peptide dimers were synthesized by using the same basic region peptide to target the same DNA sequence; one dimer was assembled with a bridged biphenyl derivative as a covalent dimerization domain, and two other dimers were assembled with the cyclodextrin guest noncovalent dimerization domains. One of the noncovalent dimers was a heterodimer that consisted of cyclodextrin and guest peptides, while the other was a homodimer that consisted of peptides bearing both cyclodextrin and the guest molecule within the same chain. Both noncovalent dimers formed the specific DNA complexes within narrower ranges of peptide concentrations and showed higher sequence selectivity than the covalent dimer did. Among the three dimers, the noncovalent homodimer that can form an intramolecular inclusion complex showed the highest sequence selectivity. Because the noncovalent homodimer with the higher stability of the circular intramolecular inclusion complex exhibited the higher sequence selectivity, it was concluded that an equilibrium involving a conformational transition of a monomeric peptide effectively reduced the stability of its nonspecific binding complex, hence increasing the efficacy of cooperative dimer formation at the specific DNA sequence.

Protein synthesis by native chemical ligation has been an effective approach for the synthesis of proteins of moderate size. The utility of this approach for protein synthesis is demonstrated by the synthesis of a transcription factor, Zif 268 that contains three zinc finger domains. This synthesis highlights the modular nature of the chemical ligation approach and the ability to synthesize, handle and fold multiple domain proteins., (Copyright 2000 John Wiley & Sons, Inc.)

It is well-known that DNA binding of native nine zinc-finger protein TEIIIA is dominated by interaction of select few fingers. Newly designed zinc-finger peptide Sp1ZF9 containing nine Cys2-His2 type motifs has been manipulated. The DNA-binding property of Sp1ZF9 was compared with those of native three zinc-finger Sp1(530-623) and artificial six zinc-finger Sp1ZF6 peptides. Although the equilibrium time was less than 0.5 h for Sp1(530-623)-DNA complex, Sp1ZF6 and Sp1ZF9 required approximately 48 and 72 h, respectively, for full complex formation. Evidently, the footprinting analysis demonstrated that Sp1ZF9 and Sp1ZF6 bind at least 27 and 18 contiguous base pairs of DNA sequence, respectively. Sp1ZF9 showed two step bindings to DNA, namely first the recognition of GC (5'-GGG-GCG-GGGCC-3') sequence by the N-terminal Sp1 domain and next the recognition of the corresponding target sequences by the middle and C-terminal Sp1 domains. In contrast with unimolecular binding of Sp1ZF9 and Sp1ZF6, two Sp1(530-623) molecules bind to one GCIII (5'-GGG-GCG-GGG-GGG-GCG-GGG-GGG++ +-GCG-GGGCC-3') site region. Semispecific complex formed at the beginning of Sp1ZF9-DNA interaction has also been characterized by kinetic analysis using surface plasmon resonance. Interestingly, the association rate constants for GC and GCIII complexes of Sp1ZF9 are smaller than those of the corresponding Sp1(530-623) complexes. Of special interest is the fact that new nine zinc-finger peptide Sp1ZF9 can bind to DNA sequence of approximately 30 base pairs. Such multi zinc-finger peptides may be useful as genome-specific transcriptional switches in future.

Using two synthetic oligonucleotides, we have constructed a new gene containing three zinc finger motifs of the Cys2-His2 type. We named this artificial gene 'Mago'. The Mago nucleotide triplets encoding the amino acid positions, described to be crucial for DNA binding specificity, have been chosen on the basis of the proposed recognition 'code' that relates the zinc finger's primary structure to the DNA binding target. Here we demonstrate that Mago protein specifically binds the 'code' DNA target, with a dissociation constant (Kd) comparable to the Kd of the well known Zif268 protein with its binding site. Moreover, we show that the deduced Mago 'code' and the 'experimental' selected DNA binding sites are almost identical, differing only in two nucleotides at the side positions.

We have designed a cationic amphipathic peptide, KALA (WEAKLAKALAKALAKHLAKALAKALKACEA), that binds to DNA, destabilizes membranes, and mediates DNA transfection. KALA undergoes a pH-dependent random coil to amphipathic alpha-helical conformational change as the pH is increased from 5.0 to 7.5. One face displays hydrophobic leucine residues, and the opposite face displays hydrophilic lysine residues. KALA-mediated release of entrapped aqueous contents from neutral and negatively charged liposomes increases with increasing helical content. KALA binds to oligonucleotides or plasmid DNA and retards their migration in gel electrophoresis. It displaces 50% of ethidium bromide from DNA at a charge ratio (+/-) of 0.9/1. In cultured cells, KALA assists oligonucleotide nuclear delivery when complexes are prepared at a 10/1 (+/-) charge ratio. KALA/DNA (10/1)(+/-) complexes mediate transfection of a variety of cell lines. The KALA sequence provides a starting point for a family of peptides that incorporate other functions to improve DNA delivery systems.

We have developed and tested a transfection compound based on synthetic peptides. It consists of a 12 amino acid DNA binding peptide (P2) with an alkyl group added to the aminoterminus (P2lip) and a peptide derived from the hemagglutinin protein (HA). The components aggregate spontaneously to particles that proved to be an efficient, easy to use and chemically stable transfection compound. With this system we found a marked correlation between transfection efficiency and mitotic activity. Cells that are allowed to perform a mitosis after exposure to either DNA-P2lip/HA or DNA-cationic liposome complexes are transfected much more efficiently than cells arrested in the cell cycle. In search of an explanation for this phenomenon we studied transport of plasmid DNA across the nuclear membrane. Plasmid DNA injected into the cytoplasm of quiescent human fibroblasts is not expressed, in contrast to DNA injected into the nucleus. FISH analysis showed that the plasmid DNA is not transported into the nucleus efficiently. Similarly, DNA-P2lip/HA complexes are readily taken up by both proliferating and nonproliferating cells, but do not readily penetrate the nuclear membrane. We conclude that delivery of plasmid DNA to the cytoplasm is not sufficient for transfection of eukaryotic cells. The nuclear membrane is apparently an important barrier. This explains why a mitotic event is required for efficient transfection with the currently available transfection systems. The implications for the further development of transfection compounds for use in vivo, where nonproliferating cells are often the target, are discussed.

Disulfide bond formation in S-acetamidomethyl (Acm) cysteine-containing peptides by successive treatments with silver trifluoromethanesulfonate (AgOTf) and dimethyl sulfoxide (DMSO)/aqueous HCl is described. An S-Acm cysteine was found to be quantitatively converted into cysteine by deprotection of the Acm group with AgOTf followed by DMSO/aqueous HCl treatment. Under these reaction conditions, no significant side reactions were observed with oxidation-sensitive amino acids such as Met, Tyr and Trp. Oxytocin and a Trp-containing peptide, urotensin II, were prepared by this method. Furthermore, regioselective two disulfide bond formation was found to be feasible by the combination of air oxidation and the AgOTf-DMSO/HCl system. This strategy has been successfully applied to the syntheses of tachyplesin I and endothelin I, which have two disulfide bonds and a Trp residue in the molecule.

The bZIP class of dimeric DNA binding proteins is characterized by a motif containing two discrete domains: a DNA contact domain defined by conserved basic and hydrophobic residues (basic domain), and a dimerization domain identified by a heptad repeat of leucine residues (zipper domain). Molecules are constructed in which the GCN4 dimerization domain is replaced by a series of stereochemically defined metal ion complexes that alter systematically the relative orientation of the basic domain peptides. Both the affinity and the specificity of DNA binding are modulated by seemingly small changes in metal complex stereochemistry. Although GCN4 binds CRE (ATGACGTCAT) and AP1 (ATGACTCAT) target sites with comparable affinity, one metallo-bZIP peptide ([G29Ts]2Fe) prefers the CRE by 4 kcal.mol-1 (1 cal = 4.184 J). Competition experiments performed with several DNAs demonstrate that discrimination between CRE and AP1 is dominated by selection against the AP1 site.

We report the complete structure determination of a 34 residue synthetic peptide with the amino acid sequence of the dimerization domain (leucine zipper) of GCN4. A high resolution structure in solution was obtained by 1H-NMR studies and distance geometry calculations followed by restrained energy minimization. A set of 20 final structures was obtained with an average root mean square deviation of 1.3 A for the backbone atoms (excluding the first and the last two residues). The structure contains an uninterrupted helix. A comparison with a structure previously determined for a larger peptide containing both the DNA-binding region (basic region) and the leucine-zipper motif shows the structural independence of the leucine-zipper domain from the contiguous DNA binding region.

In the present communication, design, synthesis and DNA binding activities of the following two peptides are reported: Dns-Gly-Ala-Gln-Lys-Leu-Ala-Cly-Lys-Val-Gly-Thr-Lys-Val-Lys-Val-Gl y-Thr-Lys-Thr - Val-OH (I) and [(H-Ala-Lys-Leu-Ala-Thr-Lys-Ala-Gly-Val-Lys-Gln-Gln-Ser-Ile-Gln-Leu-Ile- Thr- Ala-Aca-Lys-Aca)2Lys-Aca]2Lys-Val-OH (II), where Aca = NH(CH2)5CO--; Dns is a residue of 5-dimethylaminonaphtalene-1-sulfonic acid. Peptide I contains a large fraction (ca.30%) of valyl and threonyl residues, which possess a high potential for beta structure formation. Peptide II contains four repeats of the amino acid sequence present in the presumed DNA binding helix-turn-helix unit of 434 Cro repressor. These four domains are linked in such a way that two domains can interact with two halves a 14 base pair long operator site on DNA. From CD studies we have found that peptide I is in a random coil conformation in the aqueous solution in the presence of 20% trifluoroethanol. By contrast, amino acid residues of peptide II assume alpha helical, beta and random coiled conformations under the same conditions. A change in the secondary structure of the two peptides upon binding to DNA is observed. The difference CD spectra obtained by subtracting the spectra of free DNA from the spectra of peptide I--DNA complexes gives rise to a beta-like pattern. The difference CD spectra obtained for complexes of peptide II with various natural and synthetic DNAs suggest that alpha-beta-transition takes place in the presumed helix-turn-helix repeat units of peptide II upon binding to DNA. Peptide I binds more strongly to poly(dG).poly(dC) than to poly(dA).poly(dT) and poly[d(GC)].poly[d(GC)]. The binding takes place in the minor DNA groove because minor groove binding antibiotic sibiromycin can displace peptide I from a complex with poly(dG).poly(dC). Analysis of footprinting diagramms shows that peptide I specifically protects phosphodiester bonds within operator sites OR1 and OR2 of phage lambda from nuclease cleavage. By contrast, peptide II does not react specifically with operators OR1, OR2 and OR3 of phage 434 although it forms very tight complexes with DNA which are stable in the presence of 1M NH4F.

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.