Research Institute for Electronic Science, Hokkaido University, Kita-20 Nishi-10, Kita-ku, Sapporo, Hokkaido 001-0020, Japan.
ACS Chem Biol. 2010 Feb 19;5(2):215-22. doi: 10.1021/cb900263z.
Fluorescent protein (FP)-based Forster resonance energy transfer (FRET) technology is useful for development of functional indicators to visualize second messenger molecules and activation of signaling components in living cells. However, the design and construction of the functional indicators require careful optimization of their structure at the atomic level. Therefore, routine procedures for constructing FRET-based indicators currently include the adjustment of the linker length between the FPs and the sensor domain and relative dipole orientation of the FP chromophore. Here we report that, in addition to these techniques, optimization of the dimerization interface of Aequorea FPs is essential to achieve the highest possible dynamic range of signal change by FRET-based indicators. We performed spectroscopic analyses of various indicators (cameleon, TN-XL, and ATeam) and their variants. We chose variants containing mutant FPs with different dimerization properties, i.e., no, weak, or enhanced dimerization of the donor or acceptor FP. Our findings revealed that the FPs that dimerized weakly yielded high-performance FRET-based indicators with the greatest dynamic range.
荧光蛋白（FP）基的Förster 共振能量转移（FRET）技术对于开发功能指示剂以可视化活细胞中的第二信使分子和信号成分的激活非常有用。然而，功能指示剂的设计和构建需要在原子水平上仔细优化其结构。因此，目前构建基于 FRET 的指示剂的常规程序包括调整 FP 和传感器结构域之间的连接子长度以及 FP 发色团的相对偶极取向。在这里，我们报告说，除了这些技术之外，优化 Aequorea FP 的二聚化界面对于通过基于 FRET 的指示剂实现信号变化的最高可能动态范围至关重要。我们对各种指示剂（cameleon、TN-XL 和 ATeam）及其变体进行了光谱分析。我们选择了含有具有不同二聚化性质的突变 FP 的变体，即供体或受体 FP 的无、弱或增强二聚化。我们的研究结果表明，弱二聚化的 FP 产生了具有最大动态范围的高性能基于 FRET 的指示剂。
