Generation of 34S-substituted protein-bound [4Fe-4S] clusters using 34S-L-cysteine

Crack, Jason, Stewart, Melissa and Le Brun, Nicolas (2019) Generation of 34S-substituted protein-bound [4Fe-4S] clusters using 34S-L-cysteine. Biology Methods and Protocols, 4 (1). pp. 1-7. ISSN 2396-8923

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Abstract

The ability to specifically label the sulphide ions of protein-bound iron–sulphur (FeS) clusters with 34S isotope greatly facilitates structure–function studies. In particular, it provides insight when using either spectroscopic techniques that probe cluster-associated vibrations, or non-denaturing mass spectrometry, where the ∼+2 Da average increase per sulphide enables unambiguous assignment of the FeS cluster and, where relevant, its conversion/degradation products. Here, we employ a thermostable homologue of the O-acetyl-L-serine sulfhydrylase CysK to generate 34S-substituted L-cysteine and subsequently use it as a substrate for the L-cysteine desulfurase NifS to gradually supply 34S2− for in vitro FeS cluster assembly in an otherwise standard cluster reconstitution protocol.

Item Type: Article
Uncontrolled Keywords: cysteine,iron-sulphur,mass spectrometry,resonance raman spectroscopy,biochemistry, genetics and molecular biology(all),agricultural and biological sciences(all) ,/dk/atira/pure/subjectarea/asjc/1300
Faculty \ School: Faculty of Science > School of Chemistry
Related URLs:
Depositing User: LivePure Connector
Date Deposited: 11 Dec 2018 10:30
Last Modified: 22 May 2020 00:10
URI: https://ueaeprints.uea.ac.uk/id/eprint/69239
DOI: 10.1093/biomethods/bpy015

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