Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA.
J Am Chem Soc. 2010 Mar 3;132(8):2567-9. doi: 10.1021/ja9097324.
Copper, a key cofactor for many life processes, is toxic at elevated levels, and its availability is strictly controlled inside cells. Therefore, it is a challenge to visualize copper availability in the tight copper-binding environment of the cell. We report a genetically encoded fluorescent copper(I) sensor based on the copper(I)-binding-induced conformational change of a copper-responsive transcriptional regulator, Amt1. The resulting reporter, Amt1-FRET, is ratiometric, highly sensitive (K(d) = 2.5 x 10(-18) M), and selective toward copper(I). Its measured high affinity to copper(I) confirms the extremely low copper availability in yeast since Amt1 senses the upper limit of cellular copper levels in yeast and activates copper detoxification genes. Amt1-FRET operates in the dynamic range of the cellular copper buffer in mammalian cells and can report dynamic fluctuations of the cellular copper availability within minutes of perturbation. Thus, Amt1-FRET visualizes the tightly controlled copper availability in mammalian cells.
铜是许多生命过程的关键辅因子，但在高水平下具有毒性，其在细胞内的可用性受到严格控制。因此，在细胞内紧密结合铜的环境中可视化铜的可用性是一个挑战。我们报告了一种基于铜结合诱导铜反应转录调节剂 Amt1 构象变化的遗传编码荧光铜（I）传感器。由此产生的报告子 Amt1-FRET 是比率型的，对铜具有高度敏感性（K(d) = 2.5 x 10(-18) M）和选择性。其对铜（I）的高亲和力测量结果证实了酵母中铜的可用性极低，因为 Amt1 检测酵母中细胞内铜水平的上限并激活铜解毒基因。Amt1-FRET 在哺乳动物细胞的细胞铜缓冲剂的动态范围内运行，并且可以在受到干扰后的几分钟内报告细胞铜可用性的动态波动。因此，Amt1-FRET 可视化了哺乳动物细胞中受严格控制的铜可用性。
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