Effect of proline position on the antimicrobial mechanism of buforin II

Abstract

Buforin II (BF2) is a histone-derived antimicrobial peptide that causes cell death by translocating across membranes and interacting with nucleic acids. It contains one proline residue critical for its function. Previous research found that mutations replacing proline lead to decreased membrane translocation and antimicrobial activity as well as increased membrane permeabilization. This study further investigates the role of proline in BF2’s antimicrobial mechanism by considering the effect of changing proline position on membrane translocation, membrane permeabilization, and antimicrobial activity. For this purpose, four mutants were made with proline substitution (P11A) or relocation (P11A/G7P, P11A/V12P, P11A/V15P). These mutations altered the amount of α-helical content. Although antimicrobial activity correlated with the α-helical content for the peptides containing proline, membrane translocation did not. This observation suggests that factors in BF2’s bactericidal mechanism other than translocation must be altered by these mutations. To better explain these trends we also measured the nucleic acid binding and membrane permeabilization of the mutant peptides. A comparison of mutant and wild type BF2 activity revealed that BF2 relies principally on membrane translocation and nucleic acid binding for antimicrobial activity, although membrane permeabilization may play a secondary role for some BF2 variants. A better understanding of the role of proline in the BF2 antimicrobial mechanism will contribute to the further design and development of BF2 analogues. Moreover, since proline residues are prevalent among other antimicrobial peptides, this systematic characterization of BF2 provides general insights that can promote our understanding of other systems.

Investigating the nucleic acid interactions of histone‐derived antimicrobial peptides

“…For example, considering membrane translocation and DNA binding led to a more complete interpretation how proline mutants affect activity of BF2 [39]. Although AMPs are often characterized as primarily killing bacteria by either permeabilizing membranes or interacting with an intracellular component, several studies have proposed that at least some peptides may function through a combination of these different mechanisms [19, 23, 49].…”

“…A fluorescent intercalator displacement (FID) assay was used to experimentally measure the relative DNA binding of each peptide [37, 38]. The FID assay, which has been used previously to measure relative DNA binding of HDAPs [9, 39], involved loading of thiazole orange (0.55 μM), an intercalator that fluoresces upon binding double-stranded nucleic acids, in STE buffer (10 mM Tris, 50 mM NaCl, 1 mM EDTA, pH 8.0) into a quartz cuvette. Fluorescence was measured with 509 nm excitation and 527 nm emission and normalized to 0% relative fluorescence.…”

Glycin-rich antimicrobial peptide YD1 from B. amyloliquefaciens, induced morphological alteration in and showed affinity for plasmid DNA of E. coli

“…Furthermore, the positively charged residues in the α-helical sequence, and Arg-rich peptide, associate with the lipid phosphate groups to neutralize the Arg residue and allow the peptide translocation across the membrane (Amand et al 2008; Boman 1995; Su et al 2009). Recently, a report by Xie et al (2011) suggested that the position of Pro residue in buforin II was more important than the overall α-helical content for the translocation (Xie et al 2011). Consistent with these previous reports, YD1 AMP, a positively charged peptide, contains 3 glycines ([G 4,7,10 ]) and 2 proline ([P 2,8 ]) residues out of 10 amino acids.…”

Modular analysis of hipposin, a histone-derived antimicrobial peptide consisting of membrane translocating and membrane permeabilizing fragments

“…Table 1 notes how these sequences compare to the physiological sequences of hipposin, buforin I (BF1), buforin II (BF2) and parasin. Notably, the HipB region that was analogous to buforin II (BF2) included the F10W mutation that has been widely used in previous studies and shown to have analogous properties to the wildtype peptide [15, 16, 18, 20, 21]. …”