Researchers from The University of Osaka reconstruct ancestral microbial rhodopsins and show that they can be produced and experimentally tested in bacteria

Osaka, Japan – Resurrecting dinosaurs using DNA retrieved from a mosquito trapped in amber is a great movie plot, though it’s less likely to happen in the real world. However, researchers have been trying to unlock the secrets behind the evolution of a single protein family, to understand the evolution of ancestral proteins.

Now, researchers from The University of Osaka have reported a new way to bring ancient proteins back to life. The study, published in ACS Omega, has revealed that the developed methodology can help generate ancestral rhodopsins that can be tested experimentally in bacteria.

A wide range of microbes express proteins called microbial rhodopsins, which are embedded in the cell membrane and play a variety of roles, including pumping ions across the membrane or sensing light. Scientists have long wondered how members of this single family can possess such a wide array of functions, with investigations involving analyzing the protein sequences to determine their evolutionary history.

“Rhodopsins all have seven transmembrane domains that are very similar, but their extramembrane domains, which extend inside and outside of the cell, vary dramatically,” says lead author, Haruto Ishikawa. "This makes it very challenging to use standard sequence alignment techniques to trace the evolution of rhodopsin sequences from their shared ancestral proteins."

To tackle this problem, the researchers analyzed the sequences of two different microbial rhodopsins, schizorhodopsins and heliorhodopsins, using an approach that specifically accounts for insertions and deletions in the extramembrane domains. Based on this technique, they reconstructed ancestral schizorhodopsin and heliorhodopsin sequences and expressed them in bacteria.

“The results were very exciting,” explains Yasuhisa Mizutani, senior author. “Both the ancestral schizorhodopsin sequence and the ancestral heliorhodopsin sequence produced stable, mature proteins in Escherichia coli that had a distinctive color and showed characteristic spectral properties, just like existing rhodopsins.”

Similar to contemporary schizorhodopsins, the ancestral schizorhodopsin showed light-driven proton-transport activity. In contrast, the ancestral heliorhodopsin did not pump ions, consistent with current heliorhodopsins.

“Our findings show that sequence reconstruction that takes insertions and deletions into account can successfully generate full-length ancestral rhodopsins that can be experimentally produced and tested,” explains Ishikawa.
The researchers have made their analytical pipeline, ConsistASR, available for other investigators to use. The ConsistASR workflow could help reconstruct and engineer other ancestral proteins, providing functional insight into protein evolution.
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The article, “Resurrecting Full-length Ancestral Schizorhodopsins and Heliorhodopsins with Structure-guided, Indel-aware Sequence Reconstruction,” has been published in ACS Omega at https://doi.org/10.1021/acsomega.6c03010.

About The University of Osaka
The University of Osaka was founded in 1931 as one of the seven imperial universities of Japan and is now one of Japan's leading comprehensive universities with a broad disciplinary spectrum. This strength is coupled with a singular drive for innovation that extends throughout the scientific process, from fundamental research to the creation of applied technology with positive economic impacts. Its commitment to innovation has been recognized in Japan and around the world. Now, The University of Osaka is leveraging its role as a Designated National University Corporation selected by the Ministry of Education, Culture, Sports, Science and Technology to contribute to innovation for human welfare, sustainable development of society, and social transformation.
Website: https://resou.osaka-u.ac.jp/en