Dissecting the stability determinants of a challenging de novo protein fold using massively parallel design and experimentation

Design: ing 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 □ββ□ 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 □ββ□ 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 □ββ□ 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 □ββ□ 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 □ββ□ topology. Our work demonstrates how large-scale design and test cycles can solve challenging design problems while illuminating the biophysical determinants of folding. Significance Most computationally designed proteins fail to fold into their designed structures. This low success rate is a major obstacle to expanding the applications of protein design. In previous work, we discovered a small protein fold that was paradoxically challenging to design (only a 2% success rate) even though the fold itself is very simple. Here, we used a recently developed high-throughput approach to comprehensively examine the design rules for this simple fold. By designing over ten thousand proteins and experimentally measuring their folding stability, we discovered the key biophysical properties that determine the stability of these designs. Our results illustrate general lessons for protein design and also demonstrate how high-throughput stability studies can quantify the importance of different biophysical forces.