Diazirine Photoprobes for the Identification of Vancomycin-Binding Proteins

Overview

Journal ACS Bio Med Chem Au

Publisher American Chemical Society

Specialty Biochemistry

Date 2024 Apr 22

PMID 38645928

Authors

Photis Rotsides

Paula J Lee

Nakoa Webber

Kimberly C Grasty

Joris Beld

Patrick J Loll

Affiliations

Soon will be listed here.

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 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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