title: Rubredoxin refolding on nanostructured hydrophobic surfaces: evidence for a new type of biomimetic chaperones.
abstract: Rubredoxins (Rds) are small proteins containing a tetrahedral Fe(SCys)4 site. Folded forms of metal free Rds (apoRds) show greatly impaired ability to incorporate iron compared with chaotropically unfolded apoRds. In this study, formation of the Rd holoprotein (holoRd) on addition of iron to a structured, but iron-uptake incompetent apoRd was investigated in the presence of polystyrene nanoparticles (NP). In our rationale, hydrophobic contacts between apoRd and the NP surface would expose protein regions (including ligand cysteines) buried in the structured apoRd, allowing iron incorporation and folding to the native holoRd. Burial of the hydrophobic regions in the folded holoRd would allow its detachment from the NP surface. We found that both rate and yield of holoRd formation increased significantly in the presence of NP and were influenced by the NP concentration and size. Rates and yields had an optimum at "catalytic" NP concentrations (0.2 g/L NP) when using relatively small NP (46 nm diameter). At these optimal conditions, only a fraction of the apoRd was bound to the NP, consistent with the occurrence of turnover events on the NP surface. Lower rates and yields at higher NP concentrations or when using larger NP (200 nm) suggest that steric effects and molecular crowding on the NP surface favor specific "iron-uptake-competent" conformations of apoRd on the NP surface. This bio-mimetic chaperone system may be applicable to other proteins requiring an unfolding step before cofactor-triggered refolding, particularly when over-expressed under limited cofactor accessibility.
authors.affiliation: Section of Chemistry and Biomolecular Sciences, DeFENS, University of Milan, Milan, Italy.
authors.forename: Matteo
authors.initials: M
authors.lastname: Miriani
authors.forename: Stefania
authors.initials: S
authors.lastname: Iametti
authors.forename: Donald M
authors.initials: DM
authors.lastname: Kurtz
authors.forename: Francesco
authors.initials: F
authors.lastname: Bonomi
mesh_terms: D032701:Biomimetics; D005290:Ferric Compounds / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D007501:Iron / Q000378:metabolism; D007782:Lactoglobulins / Q000737:chemistry; D018832:Molecular Chaperones / Q000737:chemistry*; D049329:Nanostructures / Q000737:chemistry*; D009994:Osmolar Concentration; D011137:Polystyrenes / Q000737:chemistry; D017510:Protein Folding; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D012416:Rubredoxins / Q000737:chemistry* / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
chemical_list: D005290:Ferric Compounds; D007782:Lactoglobulins; D018832:Molecular Chaperones; D011137:Polystyrenes; D000644:Quaternary Ammonium Compounds; D012416:Rubredoxins; C077196:rubredoxin protein, Clostridium pasteurianum; D007501:Iron; C013531:ferric ammonium citrate
keywords: biomimetic chaperones; hydrophobic interfaces; latex nanoparticles; metalloproteins; protein folding; protein-interface interactions; rubredoxin