Penicillin-binding proteins (PBPs) are membrane-associated proteins involved in the biosynthesis of peptidoglycan (PG), the main component of bacterial cell walls. These proteins were discovered and named for their affinity to bind the β-lactam antibiotic penicillin. The importance of the PBPs has long been appreciated; however, specific roles of individual family members in each bacterial strain, as well as their protein-protein interactions, are yet to be understood. The apparent functional redundancy of the 4–18 PBPs that most eubacteria possess makes determination of their individual roles difficult. Existing techniques to study PBPs are not ideal because they do not directly visualize protein activity and can suffer from artifacts and perturbations of native PBP function. Therefore, development of new methods for studying the roles of individual PBPs in cell wall synthesis is required. We recently generated a library of fluorescent chemical probes containing a β-lactone scaffold that specifically targets the PBPs, enabling the visualization of their catalytic activity. Herein, we describe a general protocol to label and detect the activity of individual PBPs in Streptococcus pneumoniae using our fluorescent β-lactone probes.
|Original language||English (US)|
|Title of host publication||Methods in Enzymology|
|Editors||David M. Chenoweth|
|Publisher||Academic Press Inc.|
|Number of pages||29|
|State||Published - 2020|
|Name||Methods in Enzymology|
Bibliographical noteFunding Information:
This work was supported by the National Institutes of Health (R01 GM128439-01A1 to E.E.C. and M.E.W.), a Sloan Research Fellow Award (E.E.C.), a University of Minnesota Interdisciplinary Doctoral Fellowship (S.S.), and the University of Minnesota, Department of Chemistry. N.W.B. was supported by NIH Institutional Research and Academic Career Development Award K12 GM119955. J.D.S. was supported by the National Institutes of Health's National Center for Advancing Translational Sciences, grants TL1R002493 and UL1TR002494. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health's National Center for Advancing Translational Sciences. The DeltaVision OMX system was obtained with equipment grant NIH 1S10OD024988-01 to the Indiana University LMIC.
© 2020 Elsevier Inc.
Copyright 2020 Elsevier B.V., All rights reserved.
- Activity-based probes
- Activity-based protein profiling
- Chemical microbiology
- Chemical probe design
- Chemical probes
- Electrophilic center
- Penicillin-binding proteins
- Super-resolution fluorescence microscopy
PubMed: MeSH publication types
- Journal Article
- Research Support, N.I.H., Extramural
- Research Support, Non-U.S. Gov't