Buforin II, consisting of an N-terminal random coil, an extended helix, a hinge and a C-terminal regular a-helix, shows potent antimicrobial activity against a broad spectrum of pathogens. Therefore, to elucidate the structural features that are required for its potent antimicrobial activity and to produce buforin II analogs with increased activity, we studied the relationship between the structure and the activity of buforin II and investigated the mechanism of action of buforin II.
To elucidate the structure-activity relationship of buforin II, we synthesized a series of N- and C-terminally truncated or amino acid- substituted synthetic buforin II analogs and examined their antimicrobial activity. Deletion of the N-terminal random coil region increased slightly the antimicrobial activity, but further N-terminal truncations yielded peptide analogs with progressively decreasing activity. Removal of four amino acids from the C-terminal end of buforin II resulted in a complete loss of antimicrobial activity. The substitution of leucine for the proline-hinge decreased significantly the antimicrobial activity. Confocal fluorescence microscopic studies showed that biotinylated buforin II analogs with the proline-hinge penetrated the cell membrane and accumulated in the cytoplasm. Removal of the proline-hinge abrogated the ability of the peptide to enter cells. Biotinylated buforin II analogs without the proline-hinge localized on the cell surface and permeabilzed the cell membrane. In addition, the cell-penetrating efficiency of buforin II analogs, which varied with the a-helical content of the peptide, correlated closely with their antimicrobial potency. Our results suggest that the proline-hinge is responsible for the cell- penetrating ability of buforin II, while the penetrating- efficiency determines the antimicrobial potency of the peptide.
The mechanism of action of buforin II was studied using confocal microscopic analysis, FACS analysis and in vitro and i...