Journal
PROTEIN EXPRESSION AND PURIFICATION
Volume 73, Issue 2, Pages 161-166Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2010.05.003
Keywords
Enzymatic activity; Fluorescence; Frataxin; Iron sulfur cluster; Structure
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Funding
- MRC [U117584256]
- MRC [MC_U117584256, MC_U117533887] Funding Source: UKRI
- Medical Research Council [MC_U117584256, MC_U117533887] Funding Source: researchfish
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IscS and IscU, the two central protein components of the iron sulfur cluster assembly machinery, form a complex that is still relatively poorly characterized. In an attempt to standardize the purification of these proteins for structural studies we have developed a protocol to produce them individually in high concentration and purity. We show that IscS is a rather robust protein as long as it is produced in a PLP loaded form and that this co-factor is essential for fold stability and enzyme activity. In contrast to previous evidence, we also propose that, in contrast with previous evidence, IscU is a thermodynamically stable protein with a well defined fold but, when produced in isolation, is a 'complex-orphan protein' that is prone to unfolding if not stabilised by a co-factor or a protein partner. Our work will facilitate further structural and functional studies of these proteins and eventually lead to a better understanding of the whole machinery. (C) 2010 Elsevier Inc. All rights reserved.
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