De novo sequence redesign of a functional Ras‐binding domain globally inverted the surface charge distribution and led to extreme thermostability

To acquire extremely thermostable proteins of given functions is challenging for conventional protein engineering. Here we applied ABACUS, a statistical energy function we developed for de novo amino acid sequence design, to globally redesign a Ras‐binding domain (RBD), and obtained an extremely thermostable RBD that unfolds reversibly at above 110°C, the redesigned RBD experimentally confirmed to have expected structure and Ras‐binding interface. Directed evolution of the redesigned RBD improved its Ras‐binding affinity to the native protein level without excessive loss of thermostability. The designed amino acid substitutions were mostly at the protein surface. For many substitutions, strong epistasis or significantly differentiated effects on thermostability in the native sequence context relative to the redesigned sequence context were observed, suggesting the globally redesigned sequence to be unreachable through combining beneficial mutations of the native sequence. Further analyses revealed that by replacing 38 of a total of 48 non‐interfacial surface residues at once, ABACUS redesign was able to globally “invert” the protein's charge distribution pattern in an optimized way. Our study demonstrates that computational protein design provides powerful new tools to solve challenging protein engineering problems. The authors applied ABACUS, a statistical energy function for de novo amino acid sequence design, to globally redesign a Ras‐binding domain, and obtained an extremely thermostable RBD that unfolds reversibly at above 110°C without loss of the desired Ras‐binding activity. The analysis results show that ABACUS redesign was able to globally “invert” the protein’s charge distribution pattern in an optimized way. The study demonstrates that computational protein design provides powerful new tools to address conventionally challenging protein engineering problems.