Probing Backbone Hydrogen Bonds in Proteins by Amide‐to‐Ester Mutations

Abstract

All proteins contain characteristic backbones formed of consecutive amide bonds, which can engage in hydrogen bonds. However, the importance of these is not easily addressed by conventional technologies that only allow for side-chain substitutions. By contrast, technologies such as nonsense suppression mutagenesis and protein ligation allow for manipulation of the protein backbone. In particular, replacing the backbone amide groups with ester groups, that is, amide-to-ester mutations, is a powerful tool to examine backbone-mediated hydrogen bonds. In this minireview, we showcase examples of how amide-to-ester mutations can be used to uncover pivotal roles of backbone-mediated hydrogen bonds in protein recognition, folding, function, and structure.