Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.04.236505v1?rss=1

Authors: Adeniran, A., Tyo, K. E. J.

Abstract:

We present a reversion analysis of mutations introduced during the directed evolution of the yeast G-protein coupled receptor (GPCR) Ste2p to detect a peptide biomarker of chronic kidney disease. Two mutated receptors are analyzed in this study. Mutations acquired during directed evolution were reverted one at a time to the wild-type residue to assess the mutation's contribution to receptor function. Mutations in the first and fifth transmembrane regions, the second intracellular loop and a truncation were found to be crucial for sensitive detection of the peptide biomarker. Some mutations acquired during directed evolution were found to be neutral to or harmful for biomarker detection. Mutations were also assessed for their contributions to increasing basal activity of the evolved receptors. A similar set of crucial mutations were found in the two receptors, implying a similar mechanism detection. The mutations are reasoned to appear to give the ability to detect a smaller sized peptide, affect interaction with the G-protein and allow for prolonged signaling after stimulation. These data should provide guidance for further engineering of Ste2p and other GPCRs.

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