Engineering functional thermostable proteins using ancestral sequence reconstruction

Natural proteins are often only slightly more stable in the native state than the denatured state, and an increase in environmental temperature can easily shift the balance toward unfolding. Therefore, the engineering of proteins to improve protein stability is an area of intensive research. Thermostable proteins are required to withstand industrial process conditions, for increased shelf-life of protein therapeutics, for developing robust ‘biobricks’ for synthetic biology applications, and for research purposes (e.g., structure determination). In addition, thermostability buffers the often destabilizing effects of mutations introduced to improve other properties. Rational design approaches to engineering thermostability require structural information, but even with advanced computational methods, it is challenging to predict or parameterize all the relevant structural factors with sufficient precision to anticipate the results of a given mutation. Directed evolution is an alternative when structures are unavailable but requires extensive screening of mutant libraries. Recently, however, bioinspired approaches based on phylogenetic analyses have shown great promise. Leveraging the rapid expansion in sequence data and bioinformatic tools, ancestral sequence reconstruction can generate highly stable folds for novel applications in industrial chemistry, medicine, and synthetic biology. This review provides an overview of the factors important for successful inference of thermostable proteins by ancestral sequence reconstruction and what it can reveal about the determinants of stability in proteins.