HaloTag Assay Suggests Common Mechanism of E. coli Membrane Permeabilization Induced by Cationic Peptides.

ABSTRACT: Permeabilization of the Gram-negative bacterial outer membrane (OM) by antimicrobial peptides (AMPs) is the initial step enabling access of the AMP to the cytoplasmic membrane. We present a new single-cell, time-resolved fluorescence microscopy assay that reports on the permeabilization of the E. coli OM to small molecules with a time resolution of 3 s or better. When profluorophore JF₆₄₆ (702 Da) crosses the outer membrane (OM) and gains access to the periplasm, it binds to the localized HaloTag protein (34 kDa) and fluoresces in a characteristic hollow spatial pattern. Previous work used the much larger periplasmic GFP (27 kDa) probe, which reports on OM permeabilization to globular proteins. We test the assay on three cationic agents: Gellman random β-peptide copolymer MM

SUBMITTER: Yang Z 

PROVIDER: S-EPMC6097915 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature