Structure- and sequence-analysis inspired engineering of proteins for enhanced thermostability

•Targeting critical sites for stability improvement.•Consensus/ancestral sequence based protein stabilization.•Computational design of thermostability.•Loop grafting and chimeras for stabilization.

Protein engineering strategies for increasing stability can be improved by replacing random mutagenesis and high-throughput screening by approaches that include bioinformatics and computational design. Mutations can be focused on regions in the structure that are most flexible and involved in the early steps of thermal unfolding. Sequence analysis can often predict the position and nature of stabilizing mutations, and may allow the reconstruction of thermostable ancestral sequences. Various computational tools make it possible to design stabilizing features, such as hydrophobic clusters and surface charges. Different methods for designing chimeric enzymes can also support the engineering of more stable proteins without the need of high-throughput screening.