Insights into protein structure, stability and function from saturation mutagenesis

Where convenient phenotypic readouts are available, saturation mutagenesis coupled to deep sequencing provides a rapid and facile method to infer sequence determinants of protein structure, stability and function. We provide brief descriptions and currently available options for the various steps involved, and mention limitations of current implementations. We also highlight recent applications such as estimating relative stabilities and affinities of protein variants, mapping epitopes, protein model discrimination and prediction of mutant phenotypes. Most mutational scans have so far been applied to single genes and proteins. Additional methodological improvements are required to expand the scope to study intergenic epistasis and intermolecular interactions in macromolecular complexes.