Robustness of predictions of extremely thermally stable proteins in ancient organisms

A number of studies have addressed the environmental temperatures experienced by ancient life. Computational studies using a nonhomogeneous evolution model have estimated ancestral G + C contents of ribosomal RNAs and the amino acid compositions of ancestral proteins, generating hypotheses regarding the mesophilic last universal common ancestor. In contrast, our previous study computationally reconstructed ancestral amino acid sequences of nucleoside diphosphate kinases using a homogeneous model and then empirically resurrected the ancestral proteins. The thermal stabilities of these ancestral proteins were equivalent to or greater than those of extant homologous thermophilic proteins, supporting the thermophilic universal ancestor theory. In this study, we reinferred ancestral sequences using a dataset from which hyperthermophilic sequences were excluded. We also reinferred ancestral sequences using a nonhomogeneous evolution model. The newly reconstructed ancestral proteins are still thermally stable, further supporting the hypothesis that the ancient organisms contained thermally stable proteins and therefore that they were thermophilic.