Stabilization of the "open" conformer of apoIscU on the surface of polystyrene nanobeads accelerates assembly of a 2Fe2S structure

Insertion of cofactors into protein almost invariably seem to occur on a partially unfolded protein. These uptake-ready species can be any folding intermediate, from the nascent polypeptide to posttranslationally modified species, up to a mature and (almost) folded apoprotein, that could be selectively unfolded to take up the required cofactor. Uptake-ready species may be generated “in vitro” by unfolding with chaotropes, or “in vivo” by chaperones/co-chaperones systems. From a mechanistic standpoint, in both cases the apoprotein is maintained in an “open” conformation, that provides easier cofactor insertion. The “open” structure required for cofactor insertion may also form when proteins are non-covalently bound to a hydrophobic surface, such as polystyrene nanobeads1, 2. We studied whether formation of a cluster on apoIscU (as monitored through visible-range CD) was facilitated when apoIscU was adsorbed on the nanobeads surface. In these studies, iron salts and sulfide (or IscS + cysteine) were used as the source of cluster atoms. IscU is known to exist in structured and unstructured conformers, and the interconversion between conformers is apparently modulated by the interaction with other macromolecules, including donors, acceptors, and specific chaperones3,4. Catalytic amounts of polystyrene nanobeads (as low as 0.0005 % w/v, at ~50 micromolar apoIscU) increased the rate of appearance of the signature CD features of holoIscU containing a 2Fe2S cluster. The accelerating effect of the nanobeads on holoIscU formation was dependent on their concentration up to a 0.01% w/v threshold. Higher concentrations of nanobeads were less effective, indicating the relevance of molecular crowding effects to the observed events. This supports previous evidence about rate of assembly of holoIscU representing yet another possible point in the regulation of FeS protein biosynthesis. 1. Miriani et al. (2014) Rubredoxin refolding on nanostructured hydrophobic surfaces: Evidence for a new type of biomimetic chaperones. Proteins 82, 3154–3162 2. Miriani et al. (2014) Unfolding of beta-lactoglobulin on the surface of polystyrene nanoparticles: experimental and computational approaches. Proteins 82, 1272–1282 3. Bonomi et al. (2011) Facilitated transfer of IscU-[2Fe2S] clusters by chaperone-mediated ligand exchange. Biochemistry 50, 9641-9650 4. Yan et al. (2014) The scaffold protein IscU retains a structured conformation in the Fe-S cluster assembly complex. ChemBiochem 15, 1682-1686