Dissecting the stability determinants of a challenging de novo protein fold using massively parallel design and experimentation
Designing entirely new protein structures remains challenging because we do not fully understand the biophysical determinants of folding stability. Yet some protein folds are easier to design than others. Previous work identified the 43-residue αββα fold as especially challenging: the best designs had only a 2% success rate, compared to 39-87% success for other simple folds (1). This suggested the αββα fold would be a useful model system for gaining a deeper understanding of folding stability determinants and for testing new protein design methods. Here, we designed over ten thousand new αββα proteins and found over three thousand of them to fold into stable structures using a high-throughput protease-based assay. Nuclear magnetic resonance, hydrogen-deuterium exchange, circular dichroism, deep mutational scanning, and scrambled sequence control experiments indicated that our stable designs fold into their designed αββα structures with exceptional stability for their small size. Our large dataset enabled us to quantify the influence of universal stability determinants including nonpolar burial, helix capping, and buried unsatisfied polar atoms, as well as stability determinants unique to the αββα topology. Our work demonstrates how large-scale design and test cycles can solve challenging design problems while illuminating the biophysical determinants of folding.