Thermodynamic Stabilization of Human Frataxin

Recombinant proteins and antibodies are routinely used as drugs to treat prevalent diseases such as diabetes or cancer, while enzyme replacement and gene therapies are the main therapeutic intervention lines in rare diseases. In protein-based therapeutics, optimized in vivo stability is key as intrinsic denaturation and intracellular proteostatic degradation will limit potency, particularly in treatments requiring a sustained action, while clearance mechanisms may limit the amount of circulating protein. In vivo stability is ultimately correlated with the intrinsic thermodynamic stability of the biomolecule, but this is difficult to optimize because it often goes at the expense of reducing protein activity. Here, we have used in silico engineering approaches to thermodynamically stabilize human frataxin, a small mitochondrial protein that acts as an allosteric activator for the biosynthesis of Fe-S clusters, whose genetically-driven impairment results in a rare disease known as Friedreich ataxia. Specifically, we developed an efficient thermostability engineering computational approach that combines information on amino acid conservation, the Rosetta energy function, and two recent artificial intelligence tools – AlphaFold and ProteinMPNN – to produce thermodynamically stabilized variants of human frataxin. Such protein variants rescued the large destabilization exerted by well-known pathological mutations, with an increase over 20 °C in the melting temperature and a thermodynamic stabilization of more than 3 kcal·mol-1 at the physiological temperature. This stability surplus is translated into an enhanced resistance to proteolysis, while maintaining the protein fully functional. This case-study highlights the power of our combined computational approach to generate optimized variants, adequate for protein-based therapeutics. ### Competing Interest Statement The authors have declared no competing interest.