Watching an Engineered Calcium Biosensor Glow: Altered Reaction Pathways before Emission
Watching an Engineered Calcium Biosensor Glow: Altered Reaction Pathways before Emission
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观察工程钙生物传感器的发光：发射前反应途径的改变
DOI：
10.1021/acs.jpcb.8b10587
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发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Fang, Chong
中科院分区：
文献类型：
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作者：
Tachibana, Sean R.;Tang, Longteng;Zhu, Liangdong;Liu, Weimin;Wang, Yanli;Fang, Chong
生物传感器已成为生命科学中不可或缺的工具集。其中，基于荧光蛋白的生物传感器具有良好的生物相容性和可调发射特性，但其发展很大程度上依赖于反复试验。为了促进合理设计，我们实施了可调谐飞秒受激拉曼光谱，并辅以瞬态吸收和量子计算，以阐明基于绿色荧光蛋白-钙调蛋白复合物的发射比例式Ca2+生物传感器的单位点Pro377Arg突变体的工作机制。与母体蛋白和 Ca2+ 游离/结合状态的比较揭示了更多的结构不均匀性，但 Ca2+ 结合生物传感器内部的激发态质子转移 (ESPT) 反应总体更快。激发态下相关的光反应物和光产物振动模式揭示了 ESPT 期间 Ca2+ 结合状态下更多的发色团扭曲和捕获，以及母体蛋白在 Ca2+ 游离状态下很大程度上保守的发色团动力学。钙生物传感器内整个 ESPT 反应中未发现的结构动力学见解提供了重要的设计原则，通过生物工程方法维持亲水性、不太紧凑和更均匀的环境，并通过定向氢键（从发色团到周围的蛋白质残基）来提高 ESPT 效率和量子产率，同时保持光稳定性。
Biosensors have become an indispensable tool set in life sciences. Among them, fluorescent protein-based biosensors have great biocompatibility and tunable emission properties but their development is largely on trial and error. To facilitate a rational design, we implement tunable femtosecond stimulated Raman spectroscopy, aided by transient absorption and quantum calculations, to elucidate the working mechanisms of a single-site Pro377Arg mutant of an emission ratiometric Ca2+biosensor based on a green fluorescent protein–calmodulin complex. Comparisons with the parent protein and the Ca2+-free/bound states unveil more structural inhomogeneity yet an overall faster excited-state proton-transfer (ESPT) reaction inside the Ca2+-bound biosensor. The correlated photoreactant and photoproduct vibrational modes in the excited state reveal more chromophore twisting and trapping in the Ca2+-bound state during ESPT and the largely conserved chromophore dynamics in the Ca2+-free state from parent protein. The uncovered structural dynamics insights throughout an ESPT reaction inside a calcium biosensor provide important design principles in maintaining a hydrophilic, less compact, and more homogeneous environment with directional H-bonding (from the chromophore to surrounding protein residues) via bioengineering methods to improve the ESPT efficiency and quantum yield while maintaining photostability.