Diazirine Photoprobes for the Identification of Vancomycin-Binding Proteins

Vancomycin’s interactions with cellular targets drive its antimicrobial activity and also trigger expression of resistance against the antibiotic. Interaction partners for vancomycin have previously been identified using photoaffinity probes, which have proven to be useful tools for exploring vancom...

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Bibliographic Details
Published in:ACS bio & med chem au Vol. 4; no. 2; pp. 86 - 94
Main Authors: Rotsides, Photis, Lee, Paula J., Webber, Nakoa, Grasty, Kimberly C., Beld, Joris, Loll, Patrick J.
Format: Journal Article
Language:English
Published: United States American Chemical Society 17.04.2024
Subjects:
vancomycin
proteomics
diazirine
antivancomycin antibody
Western blotting
photoaffinity probes
ISSN:2694-2437, 2694-2437
Online Access:Get full text
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Summary:Vancomycin’s interactions with cellular targets drive its antimicrobial activity and also trigger expression of resistance against the antibiotic. Interaction partners for vancomycin have previously been identified using photoaffinity probes, which have proven to be useful tools for exploring vancomycin’s interactome. This work seeks to develop diazirine-based vancomycin photoprobes that display enhanced specificity and bear fewer chemical modifications as compared to previous photoprobes. Using proteins fused to vancomycin’s main cell-wall target, d-alanyl-d-alanine, we used mass spectrometry to show that these photoprobes specifically label known vancomycin-binding partners within minutes. In a complementary approach, we developed a Western-blot strategy targeting the vancomycin adduct of the photoprobes, eliminating the need for affinity tags and simplifying the analysis of photolabeling reactions. Together, the probes and identification strategy provide a novel and streamlined pipeline for identifying vancomycin-binding proteins.
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ISSN:2694-2437
2694-2437
DOI:10.1021/acsbiomedchemau.3c00067