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.
Fig. 1
Caption: When E. coli cells producing ancestral rhodopsin (Anc-SzR) were illuminated, the pH of the surrounding solution increased. This result supports that the ancestral rhodopsin absorbs light and, like extant schizorhodopsins, transports hydrogen ions (H⁺) into the cells.
Credit: Haruto Ishikawa
Fig. 2
Caption: During protein evolution, parts of an amino-acid sequence can be inserted or lost. Conventional calculations may not fully account for these sequence insertions and deletions, causing ancestral proteins to be predicted as unnaturally long. In this study, by explicitly considering these sequence changes, the researchers reconstructed ancestral proteins with more natural lengths and shapes.
Credit: Haruto Ishikawa
Notes
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.
