Antibacterial activities and conformations of synthetic α-defensin HNP-1 and analogs with one, two and three disulfide bridges

Structure and biological activities of synthetic peptides corresponding to human alpha-defensin HNP-1, AC(1)YC(2)RIPAC(3)IAGERRYGTC(4)IYQGRLWAFC(5)C(6) with the S-S connectivities: C-1-C-6, C-2-C-4, C-3-C-5, and its variants with one, two and three disulfide bridges were investigated. Oxidation of synthetic, reduced HNP-1 yielded a peptide with S-S connectivities C-1-C-3, C-2-C-4 and C-5-C-6, and not with the S-S linkages as in naturally occurring HNP-1. Selective protection of cysteine sulfhydryls was necessary for the formation of S-S bridges as in native HNP-1. Likewise, oxidation of peptide encompassing the segment from C-2 to C-5, resulted in the S-S linkages C-2-C-3 and C-4-C-5 instead of the expected linkage C-2-C-4 and C-3-C-5. Antibacterial activities were observed for all peptides, irrespective of how the S-S bridges were linked. Linear peptides without S-S bridges were inactive. Circular dichroism (CD) spectra suggest that peptides constrained by one and two S-S bridges do not form rigid beta-sheet structures in an aqueous environment. The spectrum of HNP-1 in an aqueous environment suggests the presence of a beta-hairpin conformation. In the presence of lipid vesicles, the S-S constrained peptides tend to adopt a beta-structure. Although the S-S connectivities observed in HNP-1 may be necessary for other physiological activities, such as chemotaxis, they are clearly not essential for antibacterial activity.