Brun Matthias A, Tan Kui-Thong, Nakata Eiji, Hinner Marlon J, Johnsson Kai
Institute of Chemical Sciences and Engineering, Ecole Polytechnique Federale de Lausanne (EPFL), CH-1015, Lausanne, Switzerland.
J Am Chem Soc. 2009 Apr 29;131(16):5873-84. doi: 10.1021/ja900149e.
Genetically encoded fluorescent sensor proteins offer the possibility to probe the concentration of key metabolites in living cells. The approaches currently used to generate such fluorescent sensor proteins lack generality, as they require a protein that undergoes a conformational change upon metabolite binding. Here we present an approach that overcomes this limitation. Our biosensors consist of SNAP-tag, a fluorescent protein and a metabolite-binding protein. SNAP-tag is specifically labeled with a synthetic molecule containing a ligand of the metabolite-binding protein and a fluorophore. In the labeled sensor, the metabolite of interest displaces the intramolecular ligand from the binding protein, thereby shifting the sensor protein from a closed to an open conformation. The readout is a concomitant ratiometric change in the fluorescence intensities of the fluorescent protein and the tethered fluorophore. The observed ratiometric changes compare favorably with those achieved in genetically encoded fluorescent sensor proteins. Furthermore, the modular design of our sensors permits the facile generation of ratiometric fluorescent sensors at wavelengths not covered by autofluorescent proteins. These features should allow semisynthetic fluorescent sensor proteins based on SNAP-tag to become important tools for probing previously inaccessible metabolites.
基因编码的荧光传感器蛋白为探测活细胞中关键代谢物的浓度提供了可能。目前用于生成此类荧光传感器蛋白的方法缺乏通用性，因为它们需要一种在代谢物结合时发生构象变化的蛋白质。在此，我们提出一种克服这一局限性的方法。我们的生物传感器由SNAP标签、一种荧光蛋白和一种代谢物结合蛋白组成。SNAP标签用一种含有代谢物结合蛋白的配体和一个荧光团的合成分子进行特异性标记。在标记的传感器中，目标代谢物将分子内配体从结合蛋白上置换下来，从而使传感器蛋白从封闭构象转变为开放构象。读出结果是荧光蛋白和连接的荧光团的荧光强度随之发生比例变化。观察到的比例变化与基因编码的荧光传感器蛋白所实现的变化相比具有优势。此外，我们传感器的模块化设计允许轻松生成在自发荧光蛋白未覆盖的波长处的比例荧光传感器。这些特性应使基于SNAP标签的半合成荧光传感器蛋白成为探测以前难以获取的代谢物的重要工具。
