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.

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Diselenide-crosslink substitutions in hirudin enhance folding rate and yield while reducing folding-intermediate heterogeneity — important chemistry for recombinant hirudin manufacturing.

Leech-derived molecules this paper discusses, each with a full profile in the ASH compound registry:

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