Improving folding properties of computationally designed proteins.

While the field of computational protein design has witnessed amazing progression in recent years, folding properties still constitute a significant barrier towards designing new and larger proteins. In order to assess and improve folding properties of designed proteins, we have developed a genetics-based folding assay and selection system based on the essential enzyme, orotate phosphoribosyl transferase from Escherichia coli. This system allows for both screening of candidate designs with good folding properties and genetic selection of improved designs. Thus, we identified single amino acid substitutions in two failed designs that rescued poorly folding and unstable proteins. Furthermore, when these substitutions were transferred into a well-structured design featuring a complex folding profile, the resulting protein exhibited native-like cooperative folding with significantly improved stability. In protein design, a single amino acid can make the difference between folding and misfolding, and this approach provides a useful new platform to identify and improve candidate designs.