Unraveling the Mechanisms of Oxidative Folding Using Single Molecule Force Spectroscopy

Disulfide bonds are formed as posttranslational modifications in a third of human proteins. The biological significance of disulfide formation is further underscored by its critical importance in numerous pathological processes including bacterial infection, viral assembly and protein misfolding disease. In eukaryotic cells, protein synthesis takes place in the cytosol where thioredoxin (TRX) prevents the formation of disulfides. Oxidative folding occurs mainly in the endoplasmic reticulum (ER) and is catalyzed by the thioredoxin-like oxidase Protein Disulfide Isomerase (PDI). Previous studies have suggested the engagement of PDI with unfolded substrates during ongoing ER translocation. However, the precise involvement of PDI during protein folding has remained elusive. Here we present a kinetic model for PDI activity during catalyzed oxidative folding. We introduce a method enabling, for the first time, independent kinetic measurements of folding and disulfide formation in a single protein substrate. Direct manipulation of the substrate enables initiation of oxidative folding from a well-defined extended state resembling the in vivo scenario. Our data indicate that structural folding is rate-limiting during catalyzed disulfide formation. Resolution of an intermediate enzyme-substrate complex appears necessary for folding to complete. Based on these observations, we propose the spontaneous rate of enzyme release as determinant of catalytic activity. We validate this hypothesis by showing that replacement of a single atom in TRX enables catalysis of disulfide formation, with PDI-like efficiency. Our findings reveal a universal catalytic mechanism common to disulfide reductases and oxidases, and can explain the functional diversification of a ubiquitous family of enzymes. Furthermore, the method presented here opens the possibility for highly specific functional screens for inhibitors of oxidative folding.