Molecular function limits divergent protein evolution on planetary timescales

Although the sequence and functional evolution of proteins have been studied in detail, it is currently unknown whether the requirement to continuously maintain the same molecular function imposes an effective limit on protein divergence. Our analysis shows that the mutual divergence rates of orthologous enzymes substantially decrease after ~1-2 billion years of independent evolution, with most orthologous enzymes being unlikely to diverge, at least on planetary timescales, beyond ~25% sequence identity. By experimentally characterizing the growth rates of all amino acid substitutions in the E. coli FolA protein, we delineate two stages of long-term sequence divergence with different contributions from sites with small and large fitness effects.