| 研究実績の概要 |
Modern proteins with complex structures are thought to have evolved from small and simple ancient proteins with prototype folds. How such prototype proteins emerged on the primitive earth remains enigmatic. RNA polymerases are ancient proteins found in all kingdoms of life and have a large complex structure consisting of multiple domains. We hypothesize that the double-psi beta-barrel (DPBB) domain at the core of RNA polymerase is the ancestor of modern RNA polymerases. We have designed a two-fold symmetric protein of the DPBB fold, which consists of a duplicated half DPBB sequence. The design of completely symmetrical DPBB sequences were carried out using the “reverse engineering evolution” computational protein design approach. We aligned the orthologous sequences of DPBB to generate a phylogenetic tree. The aligned sequences and constructed phylogenetic tree were used together to generate putative ancestral consensus sequences. The predicted ancestral sequences were then mapped onto a symmetrical DPBB backbone structural model and their energies were calculated. The top-scored designs were selected for experimental validation based on the Rosetta scores, RMSD from design template, predicted solubility and visual inspection. These designs have been validated by X-ray crystallography that the experimentally determined structures closely matches the computationally design models. This design provided support for the notion that modern day complex proteins could have been evolved from the gene duplication, fusion and diversification events from ancestral simpler proteins.
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