Characterisation of the yeast Nfs1/Isd11 cysteine desulphurase complex
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Small inorganic assemblies of alternating ferrous/ferric iron and sulphide ions, so-called iron–sulphur (Fe–S) clusters, are probably nature‟s most ancient prosthetic groups. These multipurpose reactive centres are biosynthesised by dedicated Fe–S cluster assembly proteins which are conserved in the mitochondria of all eukaryotes. One of the early actors in Fe–S cluster biosynthesis is a cysteine desulphurase, Nfs1, which catalyses the release of elemental sulphur from cysteine and plays a key role in its transfer to a molecular scaffold. Recent work has discovered that these reactions require the involvement of a small adaptor protein, Isd11. Isd11 belongs to the LYR family of proteins and helps stabilise Nfs1 upon binding. In this Thesis, heterologous production of soluble yeast Nfs1 on its own as well as in complex with yeast Isd11 in E. coli is presented. In the absence of Nfs1, Isd11 aggregated in the form of inclusion bodies from which the in vitro recovery of soluble protein could not be achieved. Both Nfs1 and the Nfs1/Isd11 complex were shown to be active, reflecting the correct fold of Nfs1. Biophysical approaches were used to provide new insights into the stoichiometry and structure of the Nfs1/Isd11 complex.
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