Ancestral Sequence Reconstruction as a Tool for the Elucidation of a Stepwise Evolutionary Adaptation

Ancestral sequence reconstruction (ASR) is a powerful tool to infer primordial sequences from contemporary, i.e., extant ones. An essential element of ASR is the computation of a phylogenetic tree whose leaves are the chosen extant sequences. Most often, the reconstructed sequence related to the root of this tree is of greatest interest: It represents the common ancestor (CA) of the sequences under study. If this sequence encodes a protein, one can "resurrect" the CA by means of gene synthesis technology and study biochemical properties of this extinct predecessor with the help of wet-lab experiments.However, ASR deduces also sequences for all internal nodes of the tree, and the well-considered analysis of these "intermediates" can help to elucidate evolutionary processes. Moreover, one can identify key mutations that alter proteins or protein complexes and are responsible for the differing properties of extant proteins. As an illustrative example, we describe the protocol for the rapid identification of hotspots determining the binding of the two subunits within the heteromeric complex imidazole glycerol phosphate synthase.