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.

中文翻译：

---

区分酵母膜受体定向进化中发现的关键，有益，中性和有害突变。

我们提出了酵母G蛋白偶联受体（GPCR）Ste2p定向进化过程中引入的突变的逆向分析，以检测慢性肾脏疾病的肽生物标志物。在这项研究中分析了两个突变的受体。将定向进化过程中获得的突变一次还原为野生型残基，以评估突变对受体功能的贡献。发现第一和第五跨膜区域，第二细胞内环和截短的突变对于灵敏检测肽生物标志物至关重要。发现在定向进化过程中获得的某些突变对生物标志物检测是中性的或有害的。还评估了突变对增加进化受体基础活性的贡献。在这两个受体中发现了一组相似的关键突变，这意味着相似的机制检测。突变被认为具有检测较小尺寸肽段的能力，影响与G蛋白的相互作用以及刺激后信号传导延长的能力。这些数据应为Ste2p和其他GPCR的进一步工程设计提供指导。