Membrane Pore Formation by Peptides Studied by Fluorescence Techniques.

Pore formation in cellular membranes by pathogen-derived proteins is a mechanism utilized by a set of microbes to exert their cytotoxic effect. On the other hand, the host cells have developed a defense mechanism to produce antimicrobial peptides to kill the pathogens by a similar membrane perforation mechanism. Furthermore, certain endogenous proteins or peptides kill the parent cells through membrane permeabilization. Analysis of the molecular details of membrane pore formation is often conducted using artificial systems, such as bilayer lipid membranes and synthetic peptides. This chapter describes two fluorescence-based methods to study peptide-induced membrane leakage. One method involves preparation of lipid vesicles loaded with a fluorophore (e.g., calcein or carboxyfluorescein) at a self-quenching concentration. If the externally added peptide forms relatively large pores (≥1 nm in diameter), the fluorophore leaks out and undergoes dequenching, resulting in a time-dependent increase in fluorescence. The other method is designed to monitor smaller pores (<1 nm in diameter). It involves the preparation of vesicles in a Ca