Figure 1. General approach to investigate the evolution of a protein-protein interaction involving intrinsically disordered domains.
Multiple sequence alignment forms the basis for the phylogeny, which is used to predict ancient variants of two interacting protein domains, CID and NCBD, respectively. The ancient variants are then resurrected by expression in Escherichia coli and purified to homogeneity. Finally, the resurrected as well as present-day variants of CID and NCBD are subjected to biophysical and computational characterization to assess the evolution of structure-function relationships.