Diselenide crosslinks for enhanced and simplified oxidative protein folding

The in vitro oxidative folding of proteins has been studied for over sixty years, providing critical insight into protein folding mechanisms. Hirudin, the most potent natural inhibitor of thrombin, is a 65-residue protein with three disulfide bonds, and is viewed as a folding model for a wide range of disulfide-rich proteins. Hirudin's folding pathway is notorious for its highly heterogeneous intermediates and scrambled isomers, limiting its folding rate and yield in vitro. Aiming to overcome these limitations, we undertake systematic investigation of diselenide bridges at native and non-native positions and investigate their effect on hirudin's folding, structure and activity. Our studies demonstrate that, regardless of the specific positions of these substitutions, the diselenide crosslinks enhanced the folding rate and yield of the corresponding hirudin analogues, while reducing the complexity and heterogeneity of the process. Moreover, crystal structure analysis confirms that the diselenide substitutions maintained the overall three-dimensional structure of the protein and left its function virtually unchanged. The choice of hirudin as a study model has implications beyond its specific folding mechanism, demonstrating the high potential of diselenide substitutions in the design, preparation and characterization of disulfide-rich proteins. Hirudin is a widely studied model for folding of disulfide-rich proteins, which folds through a slow pathway with highly heterogeneous intermediates and scrambled isomers before it reaches its native state. Here the effect of native and non-native diselenide bridges on the kinetics, yield, and heterogeneity of hirudin folding are systematically explored.

Automated summary

Hirudin, a protein with three disulfide bonds, is commonly used as a model for studying the folding of disulfide-rich proteins. This study investigates the impact of diselenide bridges at different positions on the folding kinetics, yield, and heterogeneity of hirudin, demonstrating enhanced folding rate and yield while reducing complexity. The findings suggest that diselenide substitutions could be a useful tool in designing and characterizing disulfide-rich proteins.