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: Postprint online available 08/2025
as soon as is submitted to ZB.
A photocrosslinking probe to capture the substrates of caseinolytic protease P.
Angew. Chem.-Int. Edit., DOI: 10.1002/anie.202409220 (2024)
Protein homeostasis in bacteria is regulated by proteases such as the tetradecameric caseinolytic protease P (ClpP). Although substrates of ClpP have been successfully deciphered in genetically engineered cells, methods which directly trap processed proteins within native cells remain elusive. Here, we introduce an in situ trapping strategy which utilizes trifunctional probes that bind to the active site serine of ClpP and capture adjacent substrates with an attached photocrosslinking moiety. After enrichment using an alkyne handle, substrate deconvolution by mass spectrometry (MS) is performed. We show that our two traps bind substoichiometrically to ClpP, retain protease activity, exhibit unprecedented selectivity for Staphylococcus aureus ClpP in living cells and capture numerous known and novel substrates. The exemplary validation of trapped hits using a targeted proteomics approach confirmed the fidelity of this technology. In conclusion, we provide a novel chemical platform suited for the discovery of serine protease substrates beyond genetic engineering.
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Publication type
Article: Journal article
Document type
Scientific Article
Keywords
Caseinolytic Protease P (clpp) ; Chemical Proteomics ; Chemical Biology ; Substrate Traps ; Targeted Proteomics
ISSN (print) / ISBN
1433-7851
e-ISSN
1521-3773
Publisher
Wiley
Publishing Place
Weinheim
Non-patent literature
Publications
Reviewing status
Peer reviewed
Institute(s)
CF Metabolomics & Proteomics (CF-MPC)