Abstract
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) |
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Title of host publication | Chemical Tools for Imaging, Manipulating, and Tracking Biological Systems |
Subtitle of host publication | Diverse Methods for Prokaryotic and Eukaryotic Systems |
Editors | David M. Chenoweth |
Publisher | Academic Press Inc. |
Pages | 27-55 |
Number of pages | 29 |
ISBN (Print) | 9780128201459 |
DOIs | |
State | Published - 2020 |
Publication series
Name | Methods in Enzymology |
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Volume | 638 |
ISSN (Print) | 0076-6879 |
ISSN (Electronic) | 1557-7988 |
Bibliographical note
Publisher Copyright:© 2020 Elsevier Inc.
Keywords
- Activity-based probes
- Activity-based protein profiling
- Chemical microbiology
- Chemical probe design
- Chemical probes
- Electrophilic center
- Penicillin-binding proteins
- Super-resolution fluorescence microscopy
- β-Lactones