Harnessing selenocysteine reactivity for oxidative protein folding

Abstract

Although oxidative folding of disulfide-rich proteins is often sluggish, this process can be significantly enhanced by targeted replacement of cysteines with selenocysteines. In this study, we examined the effects of a selenosulfide and native versus nonnative diselenides on the folding rates and mechanism of bovine pancreatic trypsin inhibitor. Our results show that such sulfur-to-selenium substitutions alter the distribution of key folding intermediates and enhance their rates of interconversion in a context-dependent manner.

Fig. 1. Structure of BPTI. The folded protein contains three native disulfide bonds between cysteines 5–55, 14–38, and 30–51 (PDB entry: 1BPI).

Fig. 2. Folding mechanisms of BPTI analogs. (a) Kinetically favored folding pathway for wt BPTI. R refers to the fully reduced protein; intermediates that accumulate during folding are indicated by the disulfide bonds they contain. Initial oxidation of R affords a broad distribution of single disulfide intermediates (dotted lines) that rearrange to [5–55] and [30–51].^– Qualitative estimates of the relative rates of individual steps are indicated. (b) C5U/C14U BPTI can fold via the normal BPTI mechanism (black) or by a new reaction channel (red) that avoids the long-lived N* and N′ intermediates; (c) The observation of N* suggests that C5U BPTI folds by the standard BPTI pathway, but the presence of selenocysteine increases the reactivity of both N* and N′; (d) The folding pathway for C14U/C38U BPTI is similar to that of wt BPTI, but the reactivity of the 14–38 diselenide promotes extensive precipitation.

Fig. 3. Anaerobic folding of BPTI variants at pH 8.7. Representative HPLC chromatograms of acid-quenched aliquots from folding reactions with (a) C5U BPTI with GSSG, (b) wt BPTI with GSSG; (c) C5U BPTI with GSeSeG; (d) wt BPTI with GSeSeG; and (e) C14U/C38U BPTI with GSSG. (f) Kinetic traces of the initial stages of the folding reactions of wt BPTI (blue) and C5U BPTI (red), in the presence of GSSG (dashed) or GSeSeG (solid). [protein] = 30 μM; [GSSG], [GSeSeG] = 150 μM.