Replacement of aromatic amino acids based on their consensus temporal order suggests that ancient proteins tend to be intrinsically disordered

The consensus temporal order of amino acids revealed that aromatic amino acids (F, Y and W) appear late in evolution. Interestingly, modern proteins that contains less aromatic amino acids show higher potential of intrinsically disordered properties. In this paper, we examined the hypothesis that ancient proteins with no aromatic amino acids are intrinsically disordered. Four Dephospho‐CoA Kinases (DPCK wild type, containing 5 to 19 aromatic amino acids) from 4 lower organisms were selected. All aromatics amino acids of these DPCKs were replaced with Leucine (DPCK L mutant, mimicking their ancestors) by whole gene synthesis and proteins were further purified by cobalt‐resin. The wild types were all soluble, while two of L mutants were only soluble with 4 M urea, suggesting these mutants are structurally unstable. The other two L mutants that are soluble also exhibited higher sensitivity to trypsin digestion and altered UV spectrum features of α‐helix and β‐sheet. Our results indicated that ancient proteins with less aromatic amino acids tend to be more intrinsically disordered. There are several advantages of this experimental observation. For example, intrinsically disordered proteins have less structure constraints and are more flexible to their binding partners. The impacts of these replacements to their kinase activity and specificity are under investigation.