Rubredoxin refolding on solid nanostructured hydrophobic surfaces: perspectives for developing biomimetic chaperones

Rubredoxins (Rds) are small (~54-residue) non-heme iron electron transfer proteins, with a tetrahedral Fe(SCys)4 site surrounded by a pair of iron-ligating CXXC loops1. Rds are ideal for folding studies as they: 1) are unable to incorporate metals when in their (folded) apoprotein form; 2) undergo quantitative refolding when metals are added to their chaotrope-unfolded apoprotein forms; 3) generate characteristic spectroscopic signals related to individual steps of the unfolding/refolding processes. Previous works described the metal-dependent refolding process of Rds and the influence of various denaturing agents on the process2,3. In this work we investigate the folding of Rd from Clostridium pastuerianum when adding iron to a folded, but iron-uptake incompetent apoprotein in the presence of polystyrene nanoparticles (NP) as \u201cdenaturing\u201d agents. Our rationale was that hydrophobic contacts with the NP's surface would expose protein regions (including ligand cysteines) making them capable of capturing added metals, thereby triggering refolding to the native protein structure and detachment from the NP. We found that rate and yield of refolding increase significantly in the presence of NP. Consistent with our hypothesis, t refolding rates and yields were influenced by the concentration of NP. Whereas the yield of refolded Rd increased as a function of the NP concentration, refolding rates decreased at high NP concentrations. This bio-mimetic chaperone system may be applicable to other proteins requiring a separate unfolding step prior to cofactor-triggered refolding, particularly when over-expressed in non-native hosts with limited cofactor accessibility