Journal article
Single-vesicle detection and analysis of peptide-induced membrane permeabilization
Department of Micro- and Nanotechnology, Technical University of Denmark1
Colloids and Biological Interfaces, Department of Micro- and Nanotechnology, Technical University of Denmark2
Center for Nanomedicine and Theranostics, Centers, Technical University of Denmark3
Department of Chemical and Biochemical Engineering, Technical University of Denmark4
CAPEC-PROCESS, Department of Chemical and Biochemical Engineering, Technical University of Denmark5
Department of Chemistry, Technical University of Denmark6
Physical and Biophysical Chemistry, Department of Chemistry, Technical University of Denmark7
The capability of membrane-active peptides to disrupt phospholipid membranes is often studied by investigating peptide-induced leakage of quenched fluorescent molecules from large unilamellar lipid vesicles. In this article, we explore two fluorescence microscopy-based single-vesicle detection methods as alternatives to the quenching-based assays for studying peptide-induced leakage from large unilamellar lipid vesicles.
Specifically, we use fluorescence correlation spectroscopy (FCS) to study the leakage of fluorescent molecules of different sizes from large unilamellar lipid vesicles dispersed in aqueous solution, and we use confocal imaging of surface-immobilized large unilamellar lipid vesicles to investigate whether there are heterogeneities in leakage between individual vesicles.
Of importance, we design an experimental protocol that allows us to quantitatively correlate the results of the two methods; accordingly, it can be assumed that the two methods provide complementary information about the same leakage process. We use the two methods to investigate the membrane-permeabilizing activities of three well-studied cationic membrane-active peptides: mastoparan X, melittin, and magainin 2.
The FCS results show that leakage induced by magainin 2 is less dependent on the size of the encapsulated fluorescent molecules than leakage induced by mastoparan X and melittin. The confocal imaging results show that all three peptides induce leakage by a heterogeneous process in which one portion of the vesicles are completely emptied of their contents but another portion of the vesicles are only partially emptied.
These pieces of information regarding leakage induced by mastoparan X, melittin, and magainin 2 could not readily have been obtained by the established assays for studying peptide-induced leakage from lipid vesicles.
Language: | English |
---|---|
Year: | 2015 |
Pages: | 2472-2483 |
ISSN: | 15205827 and 07437463 |
Types: | Journal article |
DOI: | 10.1021/la504752u |
ORCIDs: | Kristensen, Kasper , Ehrlich, Nicky , Henriksen, Jonas Rosager and Andresen, Thomas Lars |