Diselenide crosslinks for enhanced and simplified oxidative protein folding

The *in vitro* oxidative folding of proteins is often a slow and inefficient process, particularly for disulfide-rich proteins with complex folding pathways. This study investigates the effect of diselenide bridges on the oxidative folding of hirudin, a potent thrombin inhibitor with a notoriously heterogeneous folding pathway. By systematically introducing diselenide crosslinks at both native and non-native positions within hirudin, the researchers demonstrated that diselenide substitutions enhanced the folding rate and yield, while simultaneously reducing the complexity and heterogeneity of the process. Crystal structure analysis confirmed that the diselenide substitutions largely preserved the three-dimensional structure and biological activity of the protein. These findings highlight the potential of diselenide substitutions for improving the design, preparation, and characterization of disulfide-rich proteins.

Reem Mousa, Taghreed Hidmi, Sergei Pomyalov, Shifra Lansky, Lareen Khouri, Deborah E. Shalev, Gil Shoham, Norman Metanis