Supplementary material from "Exploring a chemical scaffold for rapid and selective photoaffinity labelling of non-ribosomal peptide synthetases in living bacterial cells"
Posted on 2022-11-24 - 09:48
Non-ribosomal peptide synthetases (NRPSs) biosynthesize many pharmaceuticals and virulence factors. The biosynthesis of these natural peptide products from biosynthetic gene clusters depends on complex regulations in bacteria. However, our current knowledge of NRPSs is based on enzymological studies using full NRPS systems and/or a single NRPS domain in heterologous hosts. Chemical and/or biochemical strategies to capture the endogenous activities of NRPSs facilitate studies on NRPS cell biology in bacterial cells. Here, we describe a chemical scaffold for the rapid and selective photoaffinity labelling of NRPSs in purified systems, crude biological samples and living bacterial cells. We synthesized photoaffinity labelling probes coupled with 5′-O-N-(phenylalanyl)sulfamoyladenosine with clickable alkyl diazirine or trifluoromethyl phenyl diazirine. We found that a trifluoromethyl phenyl diazirine-based probe cross-linked the Phe-activating domain of a GrsA-NRPS with high selectivity and sensitivity at shorter ultraviolet (UV) irradiation times (less than 5 min) relative to a prototypical benzophenone-based probe. Our results demonstrated that this quick labelling protocol can prevent damage to proteins and cells caused by long UV irradiation times, providing a mild photoaffinity labelling method for biological samples.This article is part of the theme issue ‘Reactivity and mechanism in chemical and synthetic biology’.
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Ishikawa, Fumihiro; Konno, Sho; Uchiyama, Yuko; Kakeya, Hideaki; Tanabe, Genzoh (2022). Supplementary material from "Exploring a chemical scaffold for rapid and selective photoaffinity labelling of non-ribosomal peptide synthetases in living bacterial cells". The Royal Society. Collection. https://doi.org/10.6084/m9.figshare.c.6315630.v1
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