Albumin is a redox‐active crowding agent that promotes oxidative folding of cysteine‐rich peptides

Abstract

Oxidative folding that occurs in a crowded cellular milieu is characterized by multifaceted interactions that occur among nascent polypeptides and resident components of the endoplasmic reticulum (ER) lumen. Macromolecular crowding has been considered an essential factor in the folding of polypeptides, but the excluded volume effect has not been evaluated for small, disulfide-rich peptides. In the research presented, we examined how macromolecular crowding agents, such as albumin, ovalbumin, and polysaccharides, influenced the kinetics and thermodynamics of forming disulfide bonds in four model peptides of varying molecular size from 13 residues (1.4 kDa) to 58-residues (6.5 kDa): conotoxins: GI, PVIIA, r11a, and bovine pancreatic trypsin inhibitor. Our results indicate that the excluded volume effect does not significantly alter the folding rates nor equilibria for these peptides. In stark contrast, folding reactions were dramatically accelerated, when protein-based crowding agents were present at concentrations lower than those predicted to provide the excluded volume effect. Submillimolar albumin alone was as effective as glutathione in promoting the oxidative folding of GI conotoxin at concentrations typically found in the ER. To the best of our knowledge, this is the first report and quantitative characterization of oxidative folding of peptides mediated by other than thioredoxin-based protein disulfide bonds. Our work raises a possibility that concurrent secretory and ER-resident proteins may influence the oxidative folding of small, cysteine-rich peptides not as crowding agents, but as redox-active factors.

Protein Thiol Modification and Thiol Proteomics

“…Thiol oxidation leads to misfolding and the influencing of the protein function (Buczek et al, 2007). In our experiments, we have identified an oxidised cytoskeletal protein actin involved in the rearrangement of filament in the cells, leading to cellular apoptosis (Wang et al, 2010).…”

Molecular interaction of δ-conopeptide EVIA with voltage-gated Na+ channels

“…The overall fold of δ-EVIA reflects the inhibitor cysteine knot (ICK, native fold) motif found in numerous conopeptides of the O, P and I superfamilies and other toxic and inhibitory peptides. [21,43,44] Compared to other δ-conotoxins, δ-EVIA shows an extended loop-2 region. [21] This loop is of low conformational order.…”

Thiol dependent isomerization of bovine albumin

“…Cha and Kim [24] reported that the disulfide bond between the Cys 392 and Cys 438 of human albumin is redox active and supports reduction by the thioredoxine/thioredoxine reductase system. Other authors have demonstrated that at least eight disulfide bridges in BSA can be reduced after albumin incubation with glutathione at pH 8, and that albumin disulfide bonds are effective oxidizing agents in the folding of small cysteine-rich peptides [25]. All these observations, and the results obtained in our study on aging in the presence of cysteine, point to the idea that disulfide bonds in albumin, mainly the eight such bonds involved in the Cys-Cys linear sequence, may be more than inert structural motifs and play a catalytic role mediating thiol disulfide interchanges.…”