A proline-hinge alters the characteristics of the amphipathic α-helical AMPs.
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 Gln(17) and Asp(19) (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 Glu(9) (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 Pro(10) 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.
Research Center for Proteinaceous Materials (RCPM), Chosun University, Kwangju, Korea.
Jong Kook
JK
Lee
Ramamourthy
R
Gopal
Seong-Cheol
SC
Park
Hyun Sook
HS
Ko
Yangmee
Y
Kim
Kyung-Soo
KS
Hahm
Yoonkyung
Y
Park
D000595:Amino Acid Sequence; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry* / Q000494:pharmacology*; D001419:Bacteria / Q000166:cytology / Q000187:drug effects; D002176:Candida albicans / Q000166:cytology / Q000187:drug effects; D002463:Cell Membrane Permeability / Q000187:drug effects; D002686:Chitin / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D002942:Circular Dichroism; D024202:Electrophoretic Mobility Shift Assay; D005434:Flow Cytometry; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008826:Microbial Sensitivity Tests; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D010457:Peptidoglycan / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism; D011392:Proline / Q000737:chemistry*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D053835:Unilamellar Liposomes / Q000378:metabolism
D023181:Antimicrobial Cationic Peptides; D010457:Peptidoglycan; D010713:Phosphatidylcholines; D053835:Unilamellar Liposomes; D002686:Chitin; D002784:Cholesterol; D011392:Proline