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.

Structural features of myofibrillar fish protein interacting with phosphatidylcholine liposomes

“…Nanoparticles can introduce thermodynamic instability to the surrounding proteins that can induce structural changes in proteins that lead to chemical denaturation and fibril formation, where, in the case of promotion of protein fibrillation, the nanoparticles serving as a template for protein self-assembling to result in microstructures with different features determined by the outer surface layer directly involved in the interaction (Shemetov, Nabiev, & Sukhanova, 2012). In this context, Miriani et al (2014) reported that polystyrene nanoparticles enhance the formation and stability of a specific refolding-prone conformation of the small proteins rubredoxins. According with the authors, the structural changes ensuing from protein interactions with the hydrophobic surface of polystyrene nanoparticles produce a "selective" denaturation of protein regions that lead to specific protein folding events.…”

Surface Layer of Lactobacillus helveticus MIMLh5 Promotes Endocytosis by Dendritic Cells

“…The slightly greater apparent size of proteins detached from Sl-beads ( Fig. 6A, lanes 6 to 9) may be related to the extensive unfolding associated with noncovalent interactions of proteins with polystyrene nanoparticles (24,25).…”

Protein interactions in the biological assembly of iron–sulfur clusters in Escherichia coli: Molecular and mechanistic aspects of the earliest assembly steps

“…39 The highest rates of cluster formation are observed when the protein structure is maintained in a "flexible" unfolded state (the D-state) by stabilizing this particular conformation through hydrophobic interactions with the styrene surface of latex nanoparticles. 40,41 Conversely, the lowest reconstitution rates are observed when apoIscU is "locked" in a compact structured state (the S-state) when forming an equimolar complex with the co-chaperone HscB. 31 The relatively high reconstitution rates observed for the structurally stable D39A IscU relate to its stability in the holoform, 42 and are insensitive to the presence of HscB.…”

“…As for the relevance of structural flexibility to cluster assembly on IscU, the data in Figure 2 compare the time course of appearance of the signature CD signal of a 2Fe2S cluster on wild‐type and D39A apoIscU during chemical reconstitution in the presence of various components affecting the structural flexibility of apoIscU 39 . The highest rates of cluster formation are observed when the protein structure is maintained in a “flexible” unfolded state (the D‐state) by stabilizing this particular conformation through hydrophobic interactions with the styrene surface of latex nanoparticles 40,41 . Conversely, the lowest reconstitution rates are observed when apoIscU is “locked” in a compact structured state (the S‐state) when forming an equimolar complex with the co‐chaperone HscB 31 .…”