Fluorescence and Absorbance Spectroscopy Methods to Study Membrane Perturbations by Antimicrobial Host Defense Peptides

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1548)

Abstract

Antimicrobial peptides (AMPs) are currently intensely studied because of their potential as new bactericidal and bacteriostatic agents. The mechanism of action of numerous AMPs involves the permeabilization of bacterial membranes. Several methods have been developed to study peptide–membrane interactions; in particular optical spectroscopy methods are widely used. The intrinsic fluorescence properties of the Trp indole ring in Trp-containing AMPs can be exploited by measuring the fluorescence blue shift and acrylamide-induced fluorescence quenching. One important aspect of such studies is the use of distinct models of the bacterial membrane, in most cases large unilamellar vesicles (LUVs) with different, yet well-defined, phospholipid compositions. Deploying LUVs that are preloaded with fluorescent dyes, such as calcein, also allows for the study of vesicle permeabilization by AMPs. In addition, experiments using genetically engineered live Escherichia coli cells can be used to distinguish between the effects of AMPs on the outer and inner membranes of gram-negative bacteria. In combination, these methods can provide a detailed insight into the mode of action of AMPs.

Key words

Fluorescence spectroscopy Absorbance spectroscopy Antimicrobial peptides Tryptophan Escherichia coli Membrane permeabilization Large unilamellar vesicles 

Notes

Acknowledgments

Research on antimicrobial peptides has been funded by a grant from the “Novel alternatives to antibiotics” program from the Canadian Institutes of Health Research.

E. coli ML35p was kindly provided by Dr. Robert Lehrer from the David Geffen School of Medicine at UCLA, Los Angeles, CA.

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Biochemistry Research Group, Department of Biological SciencesUniversity of CalgaryCalgaryCanada

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