Novel Diazirine Photoprobes for the Identification of Vancomycin-Binding Proteins

Abstract

Vancomycins 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 vancomycins 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 vancomycins 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. For Table of Contents Only. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/545140v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1d21b8forg.highwire.dtl.DTLVardef@10f4d4borg.highwire.dtl.DTLVardef@19bb1f0org.highwire.dtl.DTLVardef@bf3dd5_HPS_FORMAT_FIGEXP M_FIG C_FIG