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Crack, Jason, Stewart, Melissa and Le Brun, Nicolas 
ORCID: https://orcid.org/0000-0001-9780-4061
(2019)
Generation of 34S-substituted protein-bound [4Fe-4S] clusters using 34S-L-cysteine.
Biology Methods and Protocols, 4 (1).
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 (former - to 2024) |
| UEA Research Groups: | Faculty of Science > Research Centres > Centre for Molecular and Structural Biochemistry Faculty of Science > Research Groups > Chemistry of Life Processes |
| Related URLs: | |
| Depositing User: | LivePure Connector |
| Date Deposited: | 11 Dec 2018 10:30 |
| Last Modified: | 18 Oct 2024 23:49 |
| URI: | https://ueaeprints.uea.ac.uk/id/eprint/69239 |
| DOI: | 10.1093/biomethods/bpy015 |
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