The need to maintain the structural and functional integrity of an evolving protein limits the range of acceptable amino-acid substitutions — but to what extent does this constrain how far homologous protein sequences can diverge? Here, sequence divergence data are used to explore the limits of protein evolution, and to conclude that ancient proteins are continuing to diverge from one another, indicating that the protein sequence universe is slowly expanding.

A comparison of more than 1,000 orthologues of diverse proteins shows that the rate of amino-acid substitution in recent evolution is an order of magnitude lower than that expected in the absence of epistasis, indicating that epistasis is pervasive throughout protein evolution.