A Proline-Hinge Alters the Characteristics of the Amphipathic α-helical AMPs

Abstract

HP (2–20) is a 19-aa, amphipathic, α-helical peptide with antimicrobial properties that was derived from the N-terminus of Helicobacter pylori ribosomal protein L1. We previously showed that increasing the net hydrophobicity of HP (2–20) by substituting Trp for Gln17 and Asp19 (Anal 3) increased the peptide's antimicrobial activity. In hydrophobic medium, Anal 3 forms an amphipathic structure consisting of an N-terminal random coil region (residues 2–5) and an extended helical region (residues 6–20). To investigate the structure-activity relationship of Anal 3, we substituted Pro for Glu9 (Anal 3-Pro) and then examined the new peptide's three-dimensional structure, antimicrobial activity and mechanism of action. Anal 3-Pro had an α-helical structure in the presence of trifluoroethanol (TFE) and sodium dodecyl sulfate (SDS). NMR spectroscopic analysis of Anal 3-Pro's tertiary structure in SDS micelles confirmed that the kink potential introduced by Pro10 was responsible for the helix distortion. We also found that Anal 3-Pro exhibited about 4 times greater antimicrobial activity than Anal 3. Fluorescence activated flow cytometry and confocal fluorescence microscopy showed that incorporating a Pro-hinge into Anal 3 markedly reduced its membrane permeability so that it accumulated in the cytoplasm without remaining in the cell membrane. To investigate the translocation mechanism, we assessed its ability to release of FITC-dextran. The result showed Anal 3-Pro created a pore <1.8 nm in diameter, which is similar to buforin II. Notably, scanning electron microscopic observation of Candida albicans revealed that Anal 3-Pro and buforin II exert similar effects on cell membranes, whereas magainin 2 exerts a different, more damaging, effect. In addition, Anal 3-Pro assumed a helix-hinge-helix structure in the presence of biological membranes and formed micropores in both bacterial and fungal membranes, through which it entered the cytoplasm and tightly bound to DNA. These results indicate that the bending region of Anal 3- Pro peptide is prerequisite for effective antibiotic activity and may facilitate easy penetration of the lipid bilayers of the cell membrane.

Improved Bioactivity of Antimicrobial Peptides by Addition of Amino‐Terminal Copper and Nickel (ATCUN) Binding Motifs

“…However, in this family of peptides, we only need to analyze up to the proline residue, since this residue would act as a hinge if the α-helix were to extend throughout all the residues. [55] As observed in Figure 8, the VIH and GGH derivatives have five hydrophobic residues on one side of the helix, whereas the DAH derivative has only four. The parent peptide, sh -buforin, has only three hydrophobic residues (not shown).…”

Mechanisms driving the antibacterial and antibiofilm properties of Hp1404 and its analogue peptides against multidrug-resistant Pseudomonas aeruginosa

“…FACS analysis showed that Hp1404 damaged membranes while Hp1404-T1e did not. Based on these results, we investigated the potential presence of intracellular peptide targets using the DNA binding assay 50 . Surprisingly, Hp1404-T1d and Hp1404-T1e interacted with DNA, suggesting that their differences with the parent peptide may imply the existence of a distinct action mechanism 51 , 52 .…”

De Novo Design and Synthesis of Ultra-Short Peptidomimetic Antibiotics Having Dual Antimicrobial and Anti-Inflammatory Activities

“…The prokaryotic selectivity of the peptides to mammalian cells was determined by measuring their hemolytic activity toward hRBCs, as described previously in our study (Table 1) [41].…”