Oregon State University, Department of Chemistry, 263 Linus Pauling Science Centre (lab), 153 Gilbert Hall (office), Corvallis, OR 97331, USA.
Phys Chem Chem Phys. 2018 May 9;20(18):12517-12526. doi: 10.1039/c8cp01907j.
Photoactivated proton transfer (PT) wire is responsible for the glow of green fluorescent protein (GFP), which is crucial for bioimaging and biomedicine. In this work, a new GFP-S65T/S205V double mutant is developed from wild-type GFP in which the PT wire is significantly modified. We implement femtosecond transient absorption (fs-TA) and femtosecond stimulated Raman spectroscopy (FSRS) to delineate the PT process in action. The excited state proton transfer proceeds on the ∼110 ps timescale, which infers that the distance of one key link (water to T203) in the PT wire of GFP-S205V is shortened by the extra S65T mutation. The rise of an imidazolinone ring deformation mode at ∼871 cm-1 in FSRS further suggests that this PT reaction is in a concerted manner. A ∼4 ps component prior to large-scale proton dissociation through the PT wire is also retrieved, indicative of some small-scale proton motions and heavy-atom rearrangement in the vicinity of the chromophore. Our work provides deep insights into the novel hybrid PT mechanism in engineered GFP and demonstrates the power of tunable FSRS methodology in tracking ultrafast photoreactions with the desirable structural specificity in physiological environments.
光致质子转移（PT）键负责绿色荧光蛋白（GFP）的发光，这对生物成像和生物医学至关重要。在这项工作中，从野生型 GFP 中开发了一种新的 GFP-S65T/S205V 双突变体，其中 PT 键得到了显著修饰。我们采用飞秒瞬态吸收（fs-TA）和飞秒受激拉曼光谱（FSRS）来描绘作用中的 PT 过程。激发态质子转移在 ∼110 ps 的时间尺度上进行，这推断 GFP-S205V 中的 PT 键的一个关键连接（水到 T203）的距离因额外的 S65T 突变而缩短。FSRS 中在 ∼871 cm-1 处出现的咪唑啉酮环变形模式的上升进一步表明，该 PT 反应是协同的。还检索到在通过 PT 键进行大规模质子离解之前的 ∼4 ps 分量，表明在生色团附近存在一些小规模的质子运动和重原子重排。我们的工作深入了解了工程 GFP 中的新型混合 PT 机制，并展示了可调 FSRS 方法在跟踪具有生理环境中所需结构特异性的超快光反应方面的强大功能。
