Circular permutation of ligand‐binding module improves dynamic range of genetically encoded FRET‐based nanosensor
Circular permutation of ligand‐binding module improves dynamic range of genetically encoded FRET‐based nanosensor
复制标题
DOI：
10.1002/pro.266
复制
发表时间：
2009-12
期刊：
影响因子：
8
通讯作者：
S. Okada;K. Ota;Takashi Ito
中科院分区：
文献类型：
--
作者：
S. Okada;K. Ota;Takashi Ito
高时空分辨率的小分子定量测量为正确理解和准确建模代谢调控提供了坚实的基础。一种有希望实现这一目标的方法是基于细菌周围质结合蛋白（PBPs）和绿色荧光蛋白（GFP）的黄色和青色变体之间的荧光共振能量转移（FRET）的FLIP（荧光指示蛋白）纳米传感器。每个FLIP都有一个特异性结合其配体的PBP模块，以诱导构象变化，导致附着在PBP的N端和C端上的两个GFP变体模块之间的FRET变化。动态范围越大，测量结果越可靠。因此，我们试图通过在PBP模块中引入一个带有铰链环缺失的圆形排列来扩大FLIP的动态范围。所有六种循环排列的PBPs，包括结构不同的I型和II型PBPs，在用于FLIP时表现出比各自原生形式更大的动态范围。值得注意的是，圆形排列使三个PBPs完全能够作为FLIP的配体结合模块发挥作用，而这些PBPs在用作其天然形式时完全没有表现出FRET变化。这些FLIPs成功地用于测定复合溶液中的氨基酸浓度，以及实时测量活酵母细胞中的氨基酸内流。因此，循环排列策略不仅可以提高每个纳米传感器的性能，还可以扩大FLIP纳米传感器技术可以测量的代谢物的范围。
Quantitative measurement of small molecules with high spatiotemporal resolution provides a solid basis for correct understanding and accurate modeling of metabolic regulation. A promising approach toward this goal is the FLIP (fluorescent indicator protein) nanosensor based on bacterial periplasmic binding proteins (PBPs) and fluorescence resonance energy transfer (FRET) between the yellow and cyan variants of green fluorescent protein (GFP). Each FLIP has a PBP module that specifically binds its ligand to induce a conformation change, leading to a change in FRET between the two GFP variant modules attached to the N‐ and C‐termini of the PBP. The larger is the dynamic range the more reliable is the measurement. Thus, we attempted to expand the dynamic range of FLIP by introducing a circular permutation with a hinge loop deletion to the PBP module. All the six circularly permutated PBPs tested, including structurally distinct Type I and Type II PBPs, showed larger dynamic ranges than their respective native forms when used for FLIP. Notably, the circular permutation made three PBPs, which totally failed to show FRET change when used as their native forms, fully capable of functioning as a ligand binding module of FLIP. These FLIPs were successfully used for the determination of amino acid concentration in complex solutions as well as real‐time measurement of amino acid influx in living yeast cells. Thus, the circular permutation strategy would not only improve the performance of each nanosensor but also expand the repertoire of metabolites that can be measured by the FLIP nanosensor technology.