Diselenide Crosslinks for Enhanced and Simplified Oxidative Protein Folding

The oxidative folding of proteins has been studied for over sixty years, providing critical insight into protein folding mechanisms. A well-known folding model for many disulfide-rich proteins is that of hirudin. Hirudin, the most potent natural inhibitor of thrombin, is a 65-residue protein with three disulfide bonds, and folds through plagued pathway that involve highly heterogeneous intermediates and scrambled isomers. The formation of scrambled species is known to limit the rate and efficiency of in vitro oxidative folding of many proteins. In the current manuscript we describe our recent work, intended to overcome the limitations of scrambled isomers formation during oxidative protein folding. In this research we deeply investigate the utility of introducing diselenide bridges at the three native disulfide crosslinks as well as at a non-native position on hirudin’s folding, structure and function. Our studies demonstrated that, regardless of the specific positions of these substitutions, the diselenide crosslinks enhanced the folding rate and yield of the hirudin analogs, while reducing the complexity and heterogeneity of the process, and reducing the formation of scrambled isomers. A parallel, equally important, objective of our study was to test if diselenide substitutions have structural and functional effects. Crystal structure analysis as well as functional studies indicated that diselenide crosslinks maintained the overall structure of the protein without causing major changes in function and structure. To substantiate these conclusions, we provide inhibition studies and high-resolution crystal structure of the wild-type hirudin and its seleno-analogs. Taken together, we believe that the choice of hirudin as the model in this study has implications beyond its specific folding mechanism, and will serve as a useful methodology for the in vitro oxidative folding of many complex disulfide-rich proteins.