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Effect of Non-natural Hydrophobic Amino Acids on the Efficacy and Properties of the Antimicrobial Peptide C18G

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Abstract

Antimicrobial peptides (AMPs) have been an area of great interest, due to the high selectivity of these molecules toward bacterial targets over host cells and the limited development of bacterial resistance to these molecules through evolution. The peptides are known to selectively bind to bacterial cell surfaces through electrostatic interactions, and subsequently, the peptides insert into the cell membrane and cause local disruptions of membrane integrity leading to cell death. Previous experiments showed that replacing the Leu residues in the AMP C18G with other naturally occurring hydrophobic residues resulted in side-chain-dependent activities. This work extends the investigation to non-natural hydrophobic amino acids and the effect on peptide activity. Minimal inhibitory concentration (MIC) results demonstrated that amino acid substitutions containing long flexible carbon chains maintained or increased antimicrobial activity compared to natural analogues. In solution, the peptide showed aggregation only with the most hydrophobic non-natural amino acid substitutions. Binding assays using Trp fluorescence confirm a binding preference for anionic lipids while quenching experiments demonstrated that the more hydrophobic peptides are more deeply buried in the anionic lipid bilayers compared to the zwitterionic bilayers. The most effective peptides at killing bacteria were also those which showed some level of disruption of bacterial membranes; however, one peptide sequence exhibited very strong activity and very low levels of red blood cell hemolysis, yielding a promising target for future development.

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Acknowledgments

The authors would like to thank Dr. Dylan Klein for assistance with hemolysis assays and Mr. Calvin Caputo for assistance in synthesis of peptides.

Funding

This work was funded in part by NIH 1R15GM094330 to G.A.C.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, Rowan University, 201 Mullica Hill Road, Glassboro, NJ, 08028, USA

    Morgan A. Hitchner, Matthew R. Necelis, Devanie Shirley & Gregory A. Caputo

  2. Department of Molecular and Cellular Biosciences, Rowan University, 201 Mullica Hill Road, Glassboro, NJ, 08028, USA

    Gregory A. Caputo

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  1. Morgan A. Hitchner
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  2. Matthew R. Necelis
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  4. Gregory A. Caputo
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Caputo participated in research design; Hitchner, Necelis, and Shirley conducted the experiments and performed data analysis; Caputo, Necelis, and Hitchner contributed to the writing of the manuscript.

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Correspondence to Gregory A. Caputo.

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Hitchner, M.A., Necelis, M.R., Shirley, D. et al. Effect of Non-natural Hydrophobic Amino Acids on the Efficacy and Properties of the Antimicrobial Peptide C18G. Probiotics & Antimicro. Prot. 13, 527–541 (2021). https://doi.org/10.1007/s12602-020-09701-3

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