Photochromic fluorescent proteins play key roles in super-resolution microscopy and optogenetics. The light-driven structural changes that modulate the fluorescence involve both trans-to-cis isomerization and proton transfer. The mechanism, timescale and relative contribution of chromophore and protein dynamics are currently not well understood. Here, the mechanism of off-to-on-state switching in dronpa is studied using femtosecond-to-millisecond time-resolved infrared spectroscopy and isotope labelling. Chromophore and protein dynamics are shown to occur on multiple timescales, from picoseconds to hundreds of microseconds. Following excitation of the trans chromophore, a ground-state primary product is formed within picoseconds. Surprisingly, the characteristic vibrational spectrum of the neutral cis isomer appears only after several tens of nanoseconds. Further fluctuations in protein structure around the neutral cis chromophore are required to form a new intermediate, which promotes the final proton-transfer reaction. These data illustrate the interplay between chromophore dynamics and the protein environment underlying fluorescent protein photochromism.

光致变色荧光蛋白在超分辨率显微镜和光遗传学中起关键作用。调节的荧光的光驱动的结构变化同时涉及反式-到-顺式异构化和质子转移。目前尚不了解生色团和蛋白质动力学的机理，时间尺度和相对贡献。在这里，使用飞秒到毫秒的时间分辨红外光谱和同位素标记研究了德龙帕中从开到关状态转换的机理。发色团和蛋白质动力学显示在多个时间范围内，从皮秒到数百微秒。在反式激发之后生色团是一种基态初级产物，在皮秒内形成。令人惊讶的是，中性顺式异构体的特征振动光谱仅在几十纳秒后才出现。需要中性顺式生色团周围蛋白质结构的进一步波动，以形成新的中间体，从而促进最终的质子转移反应。这些数据说明了生色团动力学和荧光蛋白光致变色背后​​的蛋白环境之间的相互作用。