Mitchell Joshua A, Zhang William H, Herde Michel K, Henneberger Christian, Janovjak Harald, O'Mara Megan L, Jackson Colin J
Research School of Chemistry, Australian National University, Canberra, ACT, 2601, Australia.
Institute of Cellular Neurosciences, University of Bonn Medical School, Bonn, Germany.
Methods Mol Biol. 2017;1596:89-99. doi: 10.1007/978-1-4939-6940-1_6.
Biosensors that exploit Förster resonance energy transfer (FRET) can be used to visualize biological and physiological processes and are capable of providing detailed information in both spatial and temporal dimensions. In a FRET-based biosensor, substrate binding is associated with a change in the relative positions of two fluorophores, leading to a change in FRET efficiency that may be observed in the fluorescence spectrum. As a result, their design requires a ligand-binding protein that exhibits a conformational change upon binding. However, not all ligand-binding proteins produce responsive sensors upon conjugation to fluorescent proteins or dyes, and identifying the optimum locations for the fluorophores often involves labor-intensive iterative design or high-throughput screening. Combining the genetic fusion of a fluorescent protein to the ligand-binding protein with site-specific covalent attachment of a fluorescent dye can allow fine control over the positions of the two fluorophores, allowing the construction of very sensitive sensors. This relies upon the accurate prediction of the locations of the two fluorophores in bound and unbound states. In this chapter, we describe a method for computational identification of dye-attachment sites that allows the use of cysteine modification to attach synthetic dyes that can be paired with a fluorescent protein for the purposes of creating FRET sensors.
利用福斯特共振能量转移（FRET）的生物传感器可用于可视化生物和生理过程，并且能够在空间和时间维度上提供详细信息。在基于FRET的生物传感器中，底物结合与两个荧光团相对位置的变化相关联，从而导致FRET效率的变化，这可以在荧光光谱中观察到。因此，其设计需要一种在结合时表现出构象变化的配体结合蛋白。然而，并非所有配体结合蛋白与荧光蛋白或染料偶联后都会产生响应性传感器，并且确定荧光团的最佳位置通常需要耗费大量人力的迭代设计或高通量筛选。将荧光蛋白与配体结合蛋白进行基因融合，并结合荧光染料的位点特异性共价连接，可以对两个荧光团的位置进行精细控制，从而构建非常灵敏的传感器。这依赖于准确预测两个荧光团在结合态和未结合态的位置。在本章中，我们描述了一种用于计算鉴定染料连接位点的方法，该方法允许使用半胱氨酸修饰来连接合成染料，这些染料可与荧光蛋白配对以创建FRET传感器。
