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 post-translationally 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 nanobeads. We studied whether uptake of metal cofactors in a Cys-only coordination environment was facilitated when apoproteins were adsorbed on the nanobeads surface. Rubredoxin [Fe(Cys)4] and IscU [2Fe2S(Cys)4] were used as model proteins, and appropriate cluster insertion was monitored through visible-range CD. Iron salts and sulfide were the source of cluster atoms. Catalytic amounts of polystyrene nanobeads (as low as 0.0005 % w/v, at ~50 micromolar apoprotein) increased the rate of appearance of the signature CD features of either holoprotein. The “catalytic” effect of the nanobeads was concentration dependent up to a 0.02% w/v threshold. Higher concentrations of nanobeads were less effective, indicating the relevance of molecular crowding effects. We suggest that appropriate use of polystyrene nanobeads as a “temporary unfolding” surface may help with cofactor assembly/transfer studies whenever obtaining appropriately reactive apoproteins represents a major experimental challenge.
Unfolding of apoproteins on the surface of polystyrene nanobeads affects uptake of metal cofactors / M. Miriani, F. Bonomi, D.M.K. Kurtz, S. Iametti. ((Intervento presentato al 7. convegno International Conference Biogenesis of FeS proteins tenutosi a Columbia, USA nel 2013.
|Titolo:||Unfolding of apoproteins on the surface of polystyrene nanobeads affects uptake of metal cofactors|
MIRIANI, MATTEO (Primo)
BONOMI, FRANCESCO (Secondo)
IAMETTI, STEFANIA (Ultimo)
|Data di pubblicazione:||mag-2013|
|Settore Scientifico Disciplinare:||Settore BIO/10 - Biochimica|
|Citazione:||Unfolding of apoproteins on the surface of polystyrene nanobeads affects uptake of metal cofactors / M. Miriani, F. Bonomi, D.M.K. Kurtz, S. Iametti. ((Intervento presentato al 7. convegno International Conference Biogenesis of FeS proteins tenutosi a Columbia, USA nel 2013.|
|Appare nelle tipologie:||14 - Intervento a convegno non pubblicato|