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C-terminal amidation of PMAP-23: translocation to the inner membrane of Gram-negative bacteria

Jin-Young Kim1, Seong-Cheol Park, Moon-Young Yoon

  • 1Research Center for Proteineous Materials (RCPM), Chosun University, Kwangju, Korea.

Amino Acids
|June 1, 2010
PubMed

Insights

Amidation of pig myeloid antimicrobial peptide-23 (PMAP-23) enhances its structure and cationic charge. This modification facilitates PMAP-23 translocation into bacterial inner membranes, improving antimicrobial action.

Area of Science:

  • Antimicrobial peptides
  • Molecular biology
  • Biochemistry

Background:

  • Cathelicidins, like pig myeloid antimicrobial peptide-23 (PMAP-23), exhibit potent antimicrobial activity.
  • Amidation of antimicrobial peptides' C-terminus can enhance stability and activity while reducing cytotoxicity.

Purpose of the Study:

  • To investigate the impact of C-terminal amidation on the mode of action of PMAP-23.
  • To compare the membrane interaction and antibacterial efficacy of amidated and non-amidated PMAP-23 variants.

Main Methods:

  • Circular dichroism spectroscopy to determine secondary structure in membrane-mimetic environments.
  • Liposome-based assays (calcein release) to assess membrane permeabilization.
  • SYTOX Green uptake assays to evaluate bacterial membrane integrity.
  • Tryptophan fluorescence spectroscopy to determine peptide orientation within lipid bilayers.

Main Results:

  • Both amidated (PMAP-23N) and non-amidated (PMAP-23C) forms adopted a helix-hinge-helix structure.
  • Antibacterial activity against Gram-negative bacteria and lipopolysaccharide neutralization were similar for both forms.
  • PMAP-23N oriented perpendicularly across the inner bacterial membrane, while PMAP-23C aligned parallel to the lipid bilayer.
  • Amidation increased the peptide's cationic charge and facilitated translocation into the bacterial inner membrane.

Conclusions:

  • N-terminal amidation of PMAP-23 enhances its structural stability and cationic charge.
  • Amidation promotes a distinct membrane insertion mechanism, enabling deeper translocation into the bacterial inner membrane.
  • This enhanced membrane interaction contributes to the antimicrobial efficacy of amidated PMAP-23.

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