Measuring Peptide Translocation into Large Unilamellar Vesicles

Abstract

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1. Many of these are referred to as cell-penetrating peptides, which are frequently noted for their potential as drug delivery vectors1-3. Moreover, there is increasing interest in antimicrobial peptides that operate via non-membrane lytic mechanisms4,5, particularly those that cross bacterial membranes without causing cell lysis and kill cells by interfering with intracellular processes6,7. In fact, authors have increasingly pointed out the relationship between cell-penetrating and antimicrobial peptides1,8. A firm understanding of the process of membrane translocation and the relationship between peptide structure and its ability to translocate requires effective, reproducible assays for translocation. Several groups have proposed methods to measure translocation into large unilamellar lipid vesicles (LUVs).9-13 LUVs serve as useful models for bacterial and eukaryotic cell membranes and are frequently used in peptide fluorescent studies14,15. Here, we describe our application of the method first developed by Matsuzaki and co-workers to consider antimicrobial peptides, such as magainin and buforin II16,17. In addition to providing our protocol for this method, we also present a straightforward approach to data analysis that quantifies translocation ability using this assay. The advantages of this translocation assay compared to others are that it has the potential to provide information about the rate of membrane translocation and does not require the addition of a fluorescent label, which can alter peptide properties18, to tryptophan-containing peptides. Briefly, translocation ability into lipid vesicles is measured as a function of the Foster Resonance Energy Transfer (FRET) between native tryptophan residues and dansyl phosphatidylethanolamine when proteins are associated with the external LUV membrane (Figure 1). Cell-penetrating peptides are cleaved as they encounter uninhibited trypsin encapsulated with the LUVs, leading to disassociation from the LUV membrane and a drop in FRET signal. The drop in FRET signal observed for a translocating peptide is significantly greater than that observed for the same peptide when the LUVs contain both trypsin and trypsin inhibitor, or when a peptide that does not spontaneously cross lipid membranes is exposed to trypsin-containing LUVs. This change in fluorescence provides a direct quantification of peptide translocation over time.

Modular analysis of hipposin, a histone-derived antimicrobial peptide consisting of membrane translocating and membrane permeabilizing fragments

“…Measurement of translocation of HipC into lipid vesicles was performed using the technique proposed by Matsuzaki and co-workers [16, 18] as implemented in Spinella et al [24]. Phospholipids dissolved in chloroform were obtained from Avanti Polar Lipids (Alabaster, AL).…”

Mechanistic Landscape of Membrane-Permeabilizing Peptides

“…295 Matsuzaki et al have adapted LUVs to measure peptide translocation rate with a reporter system that involves enzymatic cleavage and FRET. 286,296,297 In this assay, LUVs containing fluorescent dansyl phosphatidylethanolamine are made with encapsulated trypsin. Peptides are then added to the LUVs.…”

Role of arginine and lysine in the antimicrobial mechanism of histone‐derived antimicrobial peptides

“…The translocation assay was performed in an analogous manner to previous HDAP studies [27,[37][38][39]. To form experimental vesicles, lipids dissolved in chloroform at a 75:20:5 ratio of POPC: POPG:DNS-PE were dried and reconstituted in a 1:1 mixture by volume of 10 mM HEPES buffer and 0.4 mM trypsin in 10 mM HEPES buffer solution for a final concentration of 0.2 mM trypsin.…”