Department of Chemistry, Oregon State University, Corvallis, OR 97331; and.
Department of Chemistry, University of Alberta, Edmonton, AB T6G 2G2, Canada.
Proc Natl Acad Sci U S A. 2014 Jul 15;111(28):10191-6. doi: 10.1073/pnas.1403712111. Epub 2014 Jul 1.
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 (Ca(2+)) sensing. This study reveals that, in the absence of Ca(2+), the dominant skeletal motion is a ∼ 170 cm(-1) phenol-ring in-plane rocking that facilitates excited-state proton transfer (ESPT) with a time constant of ∼ 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 Ca(2+) 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 Ca(2+) in physiologically relevant environments.
荧光蛋白（FPs）在生物成像和推进生物医学领域发挥了关键作用。通过工程改造，将遗传编码的 FP 变体产生的多功能荧光大大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。要可视化光激发发色团的分子舞蹈，需要一种能够分辨出内在皮秒到飞秒时间尺度上原子运动的光谱技术。我们使用飞秒受激拉曼光谱来研究最近开发的 FP-钙调蛋白生物传感器 GEM-GECO1 的激发态构象动力学，用于钙离子（Ca(2+)）传感。这项研究表明，在没有 Ca(2+)的情况下，主要的骨架运动是一个约 170 cm(-1) 的酚环面内摇摆，这促进了激发态质子转移（ESPT），时间常数约为 30 ps（比野生型 GFP 慢 6 倍），达到绿色荧光状态。该运动的功能相关性得到了分子动力学模拟的证实。在 Ca(2+)结合后，这种面内摇摆运动减少，而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。定点蛋白突变体的荧光性质进一步支持了关键残基的功能作用，包括脯氨酸 377 调节氢键网络和荧光结果。这些关键的结构动力学见解将有助于生物工程的合理设计，以生成多功能、稳健和更敏感的光学传感器，用于在生理相关环境中检测 Ca(2+)。
