Clifton Ben E, Whitfield Jason H, Sanchez-Romero Inmaculada, Herde Michel K, Henneberger Christian, Janovjak Harald, Jackson Colin J
Research School of Chemistry, The Australian National University, Building 137, Sullivans Creek Road, Canberra, ACT, 2601, Australia.
Institute of Science and Technology Austria (IST Austria), Klosterneuburg, Austria.
Methods Mol Biol. 2017;1596:71-87. doi: 10.1007/978-1-4939-6940-1_5.
Small molecule biosensors based on Förster resonance energy transfer (FRET) enable small molecule signaling to be monitored with high spatial and temporal resolution in complex cellular environments. FRET sensors can be constructed by fusing a pair of fluorescent proteins to a suitable recognition domain, such as a member of the solute-binding protein (SBP) superfamily. However, naturally occurring SBPs may be unsuitable for incorporation into FRET sensors due to their low thermostability, which may preclude imaging under physiological conditions, or because the positions of their N- and C-termini may be suboptimal for fusion of fluorescent proteins, which may limit the dynamic range of the resulting sensors. Here, we show how these problems can be overcome using ancestral protein reconstruction and circular permutation. Ancestral protein reconstruction, used as a protein engineering strategy, leverages phylogenetic information to improve the thermostability of proteins, while circular permutation enables the termini of an SBP to be repositioned to maximize the dynamic range of the resulting FRET sensor. We also provide a protocol for cloning the engineered SBPs into FRET sensor constructs using Golden Gate assembly and discuss considerations for in situ characterization of the FRET sensors.
基于Förster共振能量转移（FRET）的小分子生物传感器能够在复杂的细胞环境中以高时空分辨率监测小分子信号传导。FRET传感器可以通过将一对荧光蛋白与合适的识别结构域融合来构建，比如溶质结合蛋白（SBP）超家族的成员。然而，天然存在的SBP可能由于其热稳定性低而不适用于整合到FRET传感器中，这可能会妨碍在生理条件下成像，或者因为其N端和C端的位置对于荧光蛋白融合可能不是最优的，这可能会限制所得传感器的动态范围。在这里，我们展示了如何使用祖先蛋白重建和环形排列来克服这些问题。祖先蛋白重建作为一种蛋白质工程策略，利用系统发育信息来提高蛋白质的热稳定性，而环形排列能够重新定位SBP的末端，以最大化所得FRET传感器的动态范围。我们还提供了一个使用金门组装将工程化SBP克隆到FRET传感器构建体中的方案，并讨论了FRET传感器原位表征的注意事项。
