Design and Synthesis of Sequence-Specific DNA-Binding Peptides

Abstract

Design, synthesis and DNA binding activities of two peptides containing 32 and 102 residues are reported. A nonlinear 102-residue peptide contains four modified α helix-turn-α helix motifs of 434 cro protein. These four units are linked covalently to a carboxyterminal crosslinker containing four arms each ending with an aliphatic amino group. From CD studies we have found that in aqueous buffer in the presence of 20% trifluoroethanol the peptide residues assume α-helical, β-sheet and random-coiled conformations with the a-helical content of about 16% at room temperature. Upon complex formation between peptide and DNA, a change in the peptide conformation takes place which is consistent with an α - β transition in the DNA binding α helix-turn-α helix units of the peptide. Similar conformation changes are observed upon complex formation with the synthetic operator of a linear peptide containing residues 7–37 of 434 cro repressor. Evidently, in the complex, residues present in helices α₂ and α₃ of the two helix motif form a β-hairpin which is inserted in the minor DNA groove. The last inference is supported by our observations that the two peptides can displace the minor groove-binding antibiotic distamycin A from poly(dA) · poly(dT) and synthetic operator DNA. As revealed from DNase digestion studies, the nonlinear peptide binds more strongly to a pseudooperator O_P1, located in the cro gene, than to the operator O_R 3. A difference in the specificity shown by the non-linear peptide and wild-type cro could be attributed to a flexibility of the linker chains between the DNA-binding domains in the peptide molecule as well as to a replacement of Thr-Ala in the peptide α₂-helices. Removal of two residues from the N-terminus of helix α₂ in each of the four DNA-binding domains of the peptide leads to a loss of binding specificity.