Disulfide-containing proteins are important targets for understanding protein folding mechanisms, particularly when disulfide bond formation plays a critical role in the construction of the native structure. However, studying the disulfide-coupled folding of high-molecular-weight proteins still remains difficult due to the high hydrophobicity of folding intermediates, which promotes aggregation and interferes with their separation by reversed-phase HPLC. To address this issue, we applied a novel labeling reagent designed to capture folding intermediates to a trypsin-like protease, cocoonase, as a high-molecular-weight model protein. This reagent enabled efficient isolation of folding intermediates, facilitating detailed structural analysis. We successfully determined the disulfide bond connectivity of a major folding intermediate by subjecting the labeled species to reduction, cyanylation of Cys residues, and subsequent site-specific cleavage at the cysteine residues. The results suggest that the stepwise formation of native type disulfide bonds is important for the trypsin-like proteases and that the late-determining step of the folding mechanism is the formation of the final disulfide bond. Notably, the corresponding disulfide bond is highly conserved among the trypsin family, suggesting that it has been evolutionarily retained as a form of quality control during oxidative folding. Our findings provide new insights into the folding pathway of disulfide-containing proteases and demonstrate the utility of our labeling strategy for analyzing folding intermediates in larger proteins.
Sakata et al. (2026) studied this question.