Activity-Related Conformational Changes in d,d-Carboxypeptidases Revealed by In Vivo Periplasmic Forster Resonance Energy Transfer Assay in Escherichia coli.

ABSTRACT: One of the mechanisms of β-lactam antibiotic resistance requires the activity of d,d-carboxypeptidases (d,d-CPases) involved in peptidoglycan (PG) synthesis, making them putative targets for new antibiotic development. The activity of PG-synthesizing enzymes is often correlated with their association with other proteins. The PG layer is maintained in the periplasm between the two membranes of the Gram-negative cell envelope. Because no methods existed to detect in vivo interactions in this compartment, we have developed and validated a Förster resonance energy transfer assay. Using the fluorescent-protein donor-acceptor pair mNeonGreen-mCherry, periplasmic protein interactions were detected in fixed and in living bacteria, in single samples or in plate reader 96-well format. We show

SUBMITTER: Meiresonne NY 

PROVIDER: S-EPMC5596342 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature