Excited-state structural dynamics of a dual-emission calmodulin-green fluorescent protein sensor for calcium ion imaging
Excited-state structural dynamics of a dual-emission calmodulin-green fluorescent protein sensor for calcium ion imaging
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DOI：
10.1073/pnas.1403712111
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发表时间：
2014-07-15
影响因子：
11.1
通讯作者：
Fang, Chong
中科院分区：
文献类型：
--
作者：
Oscar, Breland G.;Liu, Weimin;Fang, Chong
荧光蛋白（FP）在生物成像和推进生物医学方面发挥了关键作用。来自工程化的、遗传上可编码的FP变体的多功能荧光极大地增强了细胞成像能力，这是由蛋白质口袋内嵌入的发色团的激发态结构动力学决定的。可视化的光激发生色团的分子编排需要一种光谱技术，能够解决原子运动的内在时间尺度上的飞秒到皮秒。我们使用飞秒受激拉曼光谱研究了最近开发的FP-钙调素生物传感器，GEM-GECO 1，用于钙离子（Ca 2+）传感的激发态构象动力学。这项研究表明，在没有Ca 2+的情况下，主要的骨骼运动类似于170 cm（-1）的酚环面内摇摆，其促进激发态质子转移（ESPT），时间常数类似于30 ps（比野生型GFP慢6倍），以达到绿色荧光状态。分子动力学模拟证实了运动的功能相关性。在Ca 2+结合时，这种面内摇摆运动减小，并且来自被捕获的光激发中性发色团的蓝色发射占主导地位，因为ESPT被抑制。位点特异性蛋白质突变体的荧光特性进一步支持了包括脯氨酸377在内的关键残基在调节氢键网络和荧光结果中的功能作用。这些重要的结构动力学见解将有助于生物工程的合理设计，以产生多功能，强大，更灵敏的光学传感器，以检测生理相关环境中的Ca 2+。
Fluorescent proteins (FPs) have played a pivotal role in bioimaging and advancing biomedicine. The versatile fluorescence from engineered, genetically encodable FP variants greatly enhances cellular imaging capabilities, which are dictated by excited-state structural dynamics of the embedded chromophore inside the protein pocket. Visualization of the molecular choreography of the photoexcited chromophore requires a spectroscopic technique capable of resolving atomic motions on the intrinsic timescale of femtosecond to picosecond. We use femtosecond stimulated Raman spectroscopy to study the excited-state conformational dynamics of a recently developed FP-calmodulin biosensor, GEM-GECO1, for calcium ion (Ca2+) sensing. This study reveals that, in the absence of Ca2+, the dominant skeletal motion is a similar to 170 cm(-1) phenol-ring in-plane rocking that facilitates excited-state proton transfer (ESPT) with a time constant of similar to 30 ps (6 times slower than wild-type GFP) to reach the green fluorescent state. The functional relevance of the motion is corroborated by molecular dynamics simulations. Upon Ca2+ binding, this in-plane rocking motion diminishes, and blue emission from a trapped photoexcited neutral chromophore dominates because ESPT is inhibited. Fluorescence properties of site-specific protein mutants lend further support to functional roles of key residues including proline 377 in modulating the H-bonding network and fluorescence outcome. These crucial structural dynamics insights will aid rational design in bioengineering to generate versatile, robust, and more sensitive optical sensors to detect Ca2+ in physiologically relevant environments.