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 the ortho site 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 ~4 and 11 ps is revealed in Cl-GFP and Br-GFP, respectively, facilitating a ~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.

跟踪荧光蛋白生色团的结构动力学是实时解锁荧光机制并实现这些功能强大且用途广泛的生物成像探针的合理设计原理的关键。通过结合最近的化学生物学和超快光谱学进展，我们制备了超文件夹绿色荧光蛋白（sfGFP）及其非规范氨基酸（ncAA）衍生物，在其上具有单个氯，溴和硝基取代基。邻位定位在嵌入发色团的酚氧原子上，并使用飞秒瞬态吸收和可调飞秒激发拉曼光谱（FSRS）的集成工具集对其进行了表征，并借助振动法向模式的量子计算进行了辅助。Cl-GFP和Br-GFP分别显示了约4和11 ps的主要振动冷却时间常数，与亲本sfGFP相比，荧光量子产率提高了约30％和12％。还从瞬态吸收光谱中检索到类似的时间常数，从而证实了固有分子时间尺度上的相关电子运动和振动运动。关键的碳-卤素拉伸运动与去质子化发色团的苯酚盐环运动在大约200℃。908和890 cm ^-1Cl-GFP和Br-GFP中的Cs在ps时间尺度上的独特振动冷却过程中表现出增强的电子激发态和蓝移活性。检索到的结构动力学因生色团的靶向位点特异性卤化而改变，因此为设计新的GFP衍生物和丰富用于生命和医学的生物成像探针工具集提供了有效的手段。