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Many naturally occurring antimicrobial peptides (AMPs) are amphipathic with a β-hairpin conformation stabilized by cross-strand disulfides across the associated β-strands. Here, we show that the disulfides are not essential. Other structuring means such as better β-turns and noncovalent cross-strand interactions can, with proper design, replace the disulfides with no loss in antimicrobial activity. Our results also demonstrate that the hairpin turn region may play a role in membrane recognition for at least one member of this class, since a homodimeric turnless β-sheet analog showed no antimicrobial activity. We also examined the effects of N-terminal fatty acid adducts on AMPs. Surprisingly, the large hydrophobic carboxylic moieties examined completely eliminated the antimicrobial activity of previously active β-hairpin peptides., (© 2016 Federation of European Biochemical Societies.)

The 62 residue peptide, SSR(1-62), whose sequence corresponds to that of ribonuclease (RNase) from Sulfolobus solfataricus, and its related peptides, SSR(1-22) and SSR(10-62), were chemically synthesized and their RNase activity and DNA-binding activity were examined. The RNase activity assay using yeast RNA or tRNA(fMet) as substrate showed that the synthetic peptide SSR(1-62) did not hydrolyze yeast RNA or tRNA(fMet). These data were not consistent with previous reports that both the native peptide isolated from S. solfataricus [Fusi et al. (1993) Eur. J. Biochem. 211, 305-311] and the recombinant peptide expressed in Escherichia coli [Fusi et al. (1995) Gene 154, 99-103] were able to hydrolyze tRNA(fMet). However, the synthetic SSR(1-62) exhibited DNA-binding activity. In the presence of synthetic SSR(1-62), the cleavage of DNA (plasmid pUCRh2-4) by restriction endonuclease (EcoRI) was not observed, suggesting that synthetic SSR(1-62) bound to DNA protected DNA from its enzymatic digestion. Neither SSR(1-22) nor SSR(10-62) prevented DNA from being cleaved by a restriction enzyme. These findings strongly suggest the importance of not only the N-terminal region of SSR(1-62) but also the C-terminal region for DNA-binding. Circular dichroism spectroscopy of synthetic SSR(1-62) indicated a beta-sheet conformation, in contrast with synthetic SSR(1-22), which exhibited an unordered conformation.

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.

A 25-residue synthetic peptide corresponding to zinc finger 31 of the Xenopus protein Xfin adopts a compact, folded conformation in the presence of zinc. Complete 1H resonance assignments have been made. The peptide contains a helix, beginning as an alpha-helix and ending as a 3(10)-helix, that extends from residue 12 to 23. Several positively charged and polar residues located on this helix are likely to be involved in interactions with DNA. Residues 1-10 appear to adopt a hairpin-like structure.