Single disulfide and linear analogues corresponding to the carboxy-terminal segment of bovine β-defensin-2: effects of introducing the β-hairpin nucleating sequence D-pro-gly on antibacterial activity and biophysical properties

Abstract

Mammalian defensins (a as well as β forms) have a β-hairpin structural motif spanning approximately 20 residues at the carboxy-terminal end. We have investigated the antibacterial activity and biophysical properties of synthetic peptides corresponding to the carboxy-terminal segment of bovine βdefensin-2 (BNBD-2): VRNHVTC₁RINRGFC₂VPIRC₃PGRTRQIGTC₄FGPRIKC₅C₆RSW (positions of disulfide bonds are C₁-C₅, C₂-C₄, and C₃-C₆). The parent sequence chosen was RCPGRTRQIGTIFGPRIKCRSW (P1), which spans the carboxy-terminal region of BNBD-2. Since the dipeptide sequence D-Pro-Gly favors nucleation of β-hairpin structures even in acyclic peptides, analogues of P1 with one D-Pro-Gly at the central portion and two D-Pro-Gly segments near the N- and C-terminal ends were generated. An analogue in which GP (residues 14 and 15) in P1 was switched to PG was also synthesized. It was observed that the cyclic form as well as their linear forms exhibited antibacterial activity. Circular dichroism and theoretical studies indicated that while the β-hairpin conformation is populated, there is conformational plasticity in the cyclic and linear peptides. The mode of bacterial killing was by membrane permeabilization. The entire mammalian defensin sequence does not appear to be essential for manifestation of antibacterial activity. Hence, short peptides corresponding to the C-terminal segments of mammalian defensins could have potential as therapeutic agents.