Diazirine Photoprobes for the Identification of Vancomycin-Binding Proteins.

Autor:	Rotsides P; Department of Biochemistry & Molecular Biology and Department of Microbiology & Immunology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19102, United States., Lee PJ; Department of Biochemistry & Molecular Biology and Department of Microbiology & Immunology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19102, United States., Webber N; Department of Biochemistry & Molecular Biology and Department of Microbiology & Immunology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19102, United States., Grasty KC; Department of Biochemistry & Molecular Biology and Department of Microbiology & Immunology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19102, United States., Beld J; Department of Biochemistry & Molecular Biology and Department of Microbiology & Immunology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19102, United States., Loll PJ; Department of Biochemistry & Molecular Biology and Department of Microbiology & Immunology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19102, United States.
Jazyk:	angličtina
Zdroj:	ACS bio & med chem Au [ACS Bio Med Chem Au] 2023 Nov 20; Vol. 4 (2), pp. 86-94. Date of Electronic Publication: 2023 Nov 20 (Print Publication: 2024).
DOI:	10.1021/acsbiomedchemau.3c00067
Abstrakt:	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. Competing Interests: The authors declare no competing financial interest. (© 2023 The Authors. Published by American Chemical Society.)
Databáze:	MEDLINE
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