Large-scale in silico mutagenesis experiments reveal optimization of genetic code and codon usage for protein mutational robustness

BACKGROUND: How, and the extent to which, evolution acts on DNA and protein sequences to ensure mutational robustness and evolvability is a long-standing open question in the field of molecular evolution. We addressed this issue through the first structurome-scale computational investigation, in which we estimated the change in folding free energy upon all possible single-site mutations introduced in more than 20,000 protein structures, as well as through available experimental stability and fitness data. RESULTS: At the amino acid level, we found the protein surface to be more robust against random mutations than the core, this difference being stronger for small proteins. The destabilizing and neutral mutations are more numerous in the core and on the surface, respectively, whereas the stabilizing mutations are about 4% in both regions. At the genetic code level, we observed smallest destabilization for mutations that are due to substitutions of base III in the codon, followed by base I, bases I+III, base II, and other multiple base substitutions. This ranking highly anticorrelates with the codon-anticodon mispairing frequency in the translation process. This suggests that the standard genetic code is optimized to limit the impact of random mutations, but even more so to limit translation errors. At the codon level, both the codon usage and the usage bias appear to optimize mutational robustness and translation accuracy, especially for surface residues. CONCLUSION: Our results highlight the non-universality of mutational robustness and its multiscale dependence on protein features, the structure of the genetic code, and the codon usage. Our analyses and approach are strongly supported by available experimental mutagenesis data.