Active site center redesign increases protein stability preserving catalysis in thioredoxin.

The stabilization of natural proteins is a long-standing desired goal in protein engineering. Optimizing the hydrophobicity of the protein core often results in extensive stability enhancements. However, the presence of totally or partially buried catalytic charged residues, essential for protein function, has limited the applicability of this strategy. Here, focusing on the thioredoxin, we aimed to augment protein stability by removing buried charged residues in the active site without loss of catalytic activity. To this end, we performed a charged-to-hydrophobic substitution of a buried and functional group, resulting in a significant stability increase yet abolishing catalytic activity. Then, to simulate the catalytic role of the buried ionizable group, we designed a combinatorial library of variants targeting a set of seven surface residues adjacent to the active site. Notably, more than 50% of the library variants restored, to some extent, the catalytic activity. The combination of experimental study of 2% of the library with the prediction of the whole mutational space by partial least squares regression revealed that a single point mutation at the protein surface is sufficient to fully restore the catalytic activity without thermostability cost. As a result, we engineered one of the highest thermal stabilities reported for a protein with a natural occurring fold (137°C). Further, our hyperstable variant preserves the catalytic activity both in vitro and in vivo.