驗證高通量實驗對CueR蛋白金屬選擇特性之假說

Abstract

金屬離子在細胞內的濃度需嚴密管控，而MerR蛋白家族是細菌用來監控毒性金屬離子的轉錄因子。然而部分成員對金屬離子不具選擇性。其中，CueR是調控細胞中一價銅離子濃度的蛋白，其含有的金屬結合域與金屬離子結合會改變蛋白的構型，進而促進下游基因表達。 前人用高通量實驗產生CueR金屬結合域113、116、117和118位點的所有胺基酸組合，並在細胞中量測每個變異株受金、銀、銅離子誘導造成的基因表達，結果顯示脯胺酸 (Proline) 的出現，對基因表達量影響最顯著，然而過去常認為高通量實驗的準確性有待商榷。因此為了探討高通量實驗之準確性，我將這些變異株在細胞中進行金屬誘導表達的測試，發現脯胺酸 (Proline) 在113、118位點增加誘導基因表達量，在116、117位點則降低表達，而在蛋白與銅離子反應的體外實驗也得到相符的結果，代表金屬結合域序列的改變確實影響蛋白對金屬離子的反應，也呈現出高通量實驗之準確性，期望本研究對未來欲進行高通量實驗以及育研究金屬結合蛋白者，能夠提供積極的資訊。

The intracellular concentration of metal ions must be tightly regulated, and the MerR protein family serves as transcription factors for bacteria to monitor toxic metal ions. However, some members of this family lack selectivity for specific metal ions. Among them, CueR is a protein that regulates the concentration of monovalent copper ions in cells. Its metal-binding domain undergoes conformational changes upon binding with metal ions, thereby promoting the expression of downstream genes. Previous studies employed high-throughput experiments to generate all amino acid combinations at positions 113, 116, 117, and 118 of the CueR metal-binding domain. They measured gene expression levels induced by gold, silver, and copper ions for each variant in cells. The results indicated that the presence of proline (P) had the most significant impact on gene expression levels. However, the accuracy of high-throughput experiments has often been questioned. To evaluate the accuracy of high-throughput experiments the same time, I tested the metal-induced expression of these variants in cells. It was found that proline at CueR positions 113 and 118 increased the induction of gene expression, while at positions 116 and 117, it reduced expression. Consistent results were obtained in in vitro experiments measuring the affinity of the protein for copper ions, demonstrating that changes in the sequence of the metal-binding domain indeed affect the protein's affinity for metal ions. These findings also validate the accuracy of high-throughput experiments. It is hoped that this study will provide valuable insights for future researchers intending to conduct high-throughput experiments and study metal-binding proteins.