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Preprint
Novel Diazirine Photoprobes for the Identification of Vancomycin-Binding Proteins
bioRxiv
15 Jun 2023
Abstract
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 use 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 novel vancomycin-binding proteins.
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Details
- Title
- Novel Diazirine Photoprobes for the Identification of Vancomycin-Binding Proteins
- Creators
- Photis Rotsides - Drexel UniversityPaula J Lee - Drexel UniversityNakoa Webber - Drexel UniversityKimberly C Grasty - Drexel UniversityJoris Beld - Drexel UniversityPatrick J Loll - Drexel University
- Publication Details
- bioRxiv
- Publisher
- Cold Spring Harbor Laboratory
- Resource Type
- Preprint
- Language
- English
- Academic Unit
- Biochemistry and Molecular Biology; Microbiology and Immunology
- Other Identifier
- 991020641549604721