Investigating the Role of Proline in Buforin II Function

Buforin II (BF2) is a 21 amino acid long antimicrobial peptide. Unlike many antimicrobial peptides that induce cell death by disrupting the cellular membrane, BF2 exhibits potent antimicrobial activity without significant membrane permeabilization. A histone derivative, BF2 is hypothesized to kill bacteria by translocating across the cell membrane and binding to nucleic acids. Its membrane-penetrating property makes it a potential model for novel drug delivery systems. Pro 11 of BF2 been shown to play an important role in membrane translocation. To investigate the role played by proline, it was replaced with alanine (P11A) or reintroduced at various locations (P11A/G7P, P11A/V12P and P11A/V15P). Changing the location of the proline residue alters the peptide's overall helicity and affects the peptide's antimicrobial activity. Lipid vesicle assays showed that an optimal amount of α-helicity appears related to translocation, as increased or decreased helicity both led to reduced translocation compared to wild type BF2. However, antimicrobial activities did not correlate clearly with translocation abilities. To better understand the antimicrobial mechanism of BF2 and the role of proline, pore formation and DNA binding were investigated. The pore-forming abilities of wild type BF2 and its proline mutants were examined with a lipid vesicle dye-leakage assay. These experiments showed that increased α-helicity correlates with peptides' increased ability to cause membrane permeabilization. A fluorescent intercalator assay was used to determine the peptides' ability to bind nucleic acids. These studies revealed that while P11A/G7P exhibits a significantly stronger DNA binding ability than wild type BF2, the other mutants have similar DNA binding abilities. Together, this data helps to explain the imperfect correlation between the peptides' respective antimicrobial activities and their abilities to translocate and sheds light on the role of proline in BF2 function.