Dissecting Optical Response and Molecular Structure of Fluorescent Proteins With Non-canonical Chromophores
Dissecting Optical Response and Molecular Structure of Fluorescent Proteins With Non-canonical Chromophores
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DOI：
10.3389/fmolb.2020.00131
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发表时间：
2020-07-07
影响因子：
5
通讯作者：
Fang, Chong
中科院分区：
文献类型：
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作者：
Oscar, Breland G.;Zhu, Liangdong;Fang, Chong
跟踪荧光蛋白发色团的结构动力学是真实的解锁荧光机制并实现这些强大且多功能的生物成像探针的合理设计原则的关键。结合化学生物学和超快光谱学的最新进展，我们制备了超折叠绿色荧光蛋白（sfGFP）及其非规范氨基酸（ncAA）衍生物，这些衍生物在嵌入发色团的酚氧的邻位处具有单个氯、溴和硝基取代基，并使用飞秒瞬态吸收和可调谐飞秒受激拉曼光谱（FSRS）的集成工具集对其进行表征，借助振动正常模式的量子计算。在Cl-GFP和Br-GFP中分别揭示了类似于4和11 ps的主导振动冷却时间常数，促进了荧光量子产率相对于亲本sfGFP的类似于30%和12%的增加。类似的时间常数也检索到的瞬态吸收光谱，证实了相关的电子和振动运动的内在分子时标。关键的碳卤素伸缩运动与酚环运动的去质子化发色团在约。908和890 cm（-1）在Cl-GFP和Br-GFP中表现出增强的电子激发态活性和在ps时间尺度上明显的振动冷却过程中的蓝移。检索到的结构动力学变化，由于有针对性的位点特异性卤化的发色团，从而提供了一种有效的手段，设计新的GFP衍生物和丰富的生物成像探针工具集的生命和医学科学。
Tracking the structural dynamics of fluorescent protein chromophores holds the key to unlocking the fluorescence mechanisms in real time and enabling rational design principles of these powerful and versatile bioimaging probes. By combining recent chemical biology and ultrafast spectroscopy advances, we prepared the superfolder green fluorescent protein (sfGFP) and its non-canonical amino acid (ncAA) derivatives with a single chlorine, bromine, and nitro substituent at theorthosite to the phenolate oxygen of the embedded chromophore, and characterized them using an integrated toolset of femtosecond transient absorption and tunable femtosecond stimulated Raman spectroscopy (FSRS), aided by quantum calculations of the vibrational normal modes. A dominant vibrational cooling time constant of similar to 4 and 11 ps is revealed in Cl-GFP and Br-GFP, respectively, facilitating a similar to 30 and 12% increase of the fluorescent quantum yield vs. the parent sfGFP. Similar time constants were also retrieved from the transient absorption spectra, substantiating the correlated electronic and vibrational motions on the intrinsic molecular timescales. Key carbon-halogen stretching motions coupled with phenolate ring motions of the deprotonated chromophores at ca. 908 and 890 cm(-1)in Cl-GFP and Br-GFP exhibit enhanced activities in the electronic excited state and blue-shift during a distinct vibrational cooling process on the ps timescale. The retrieved structural dynamics change due to targeted site-specific halogenation of the chromophore thus provides an effective means to design new GFP derivatives and enrich the bioimaging probe toolset for life and medical sciences.