Effects of Hydrophobic Amino Acid Substitutions on Antimicrobial Peptide Behavior

  • Kimberly D. Saint Jean
  • Karlee D. Henderson
  • Christina L. Chrom
  • Louisa E. Abiuso
  • Lindsay M. Renn
  • Gregory A. Caputo


Antimicrobial peptides (AMPs) are naturally occurring components of the immune system that act against bacteria in a variety of organisms throughout the evolutionary hierarchy. There have been many studies focused on the activity of AMPs using biophysical and microbiological techniques; however, a clear and predictive mechanism toward determining if a peptide will exhibit antimicrobial activity is still elusive, in addition to the fact that the mechanism of action of AMPs has been shown to vary between peptides, targets, and experimental conditions. Nonetheless, the majority of AMPs contain hydrophobic amino acids to facilitate partitioning into bacterial membranes and a net cationic charge to promote selective binding to the anionic surfaces of bacteria over the zwitterionic host cell surfaces. This study explores the role of hydrophobic amino acids using the peptide C18G as a model system. These changes were evaluated for the effects on antimicrobial activity, peptide-lipid interactions using Trp fluorescence spectroscopy, peptide secondary structure formation, and bacterial membrane permeabilization. The results show that while secondary structure formation was not significantly impacted by the substitutions, antibacterial activity and binding to model lipid membranes were well correlated. The variants containing Leu or Phe as the sole hydrophobic groups bound bilayers with highest affinity and were most effective at inhibiting bacterial growth. Peptides with Ile exhibited intermediate behavior while those with Val or α-aminoisobutyric acid (Aib) showed poor binding and activity. The Leu, Phe, and Ile peptides demonstrated a clear preference for anionic bilayers, exhibiting significant emission spectrum shifts upon binding. Similarly, the Leu, Phe, and Ile peptides demonstrated greater ability to disrupt lipid vesicles and bacterial membranes. In total, the data indicate that hydrophobic moieties in the AMP sequence play a significant role in the binding and ability of the peptide to exhibit antibacterial activity.


Antimicrobial peptides Fluorescence Lipid binding C18G 



The authors would like to thank Renee Demarest at the Rowan School of Osteopathic Medicine for the assistance with the cytotoxicity assays.

Authors’ Contribution

Caputo participated in research design; Saint Jean, Henderson, Abiuso, Chrom, and Renn conducted the experiments and performed data analysis; and Caputo, Saint Jean, and Henderson contributed to the writing of the manuscript.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

12602_2017_9345_MOESM1_ESM.docx (3.1 mb)
ESM 1 (DOCX 3.07 mb)


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Copyright information

© Springer Science+Business Media, LLC 2017

Authors and Affiliations

  • Kimberly D. Saint Jean
    • 1
  • Karlee D. Henderson
    • 1
  • Christina L. Chrom
    • 1
  • Louisa E. Abiuso
    • 1
  • Lindsay M. Renn
    • 1
  • Gregory A. Caputo
    • 1
    • 2
  1. 1.Department of Chemistry and BiochemistryRowan UniversityGlassboroUSA
  2. 2.Department of Molecular and Cellular BiosciencesRowan UniversityGlassboroUSA

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