Lacombat Fabien, Plaza Pascal, Plamont Marie-Aude, Espagne Agathe
Ecole normale supérieure, PSL Research University , Département de Chimie, PASTEUR, 24 rue Lhomond, 75005 Paris, France.
Sorbonne Universités, UPMC Univ Paris 06, PASTEUR, 24 rue Lhomond, 75005 Paris, France.
J Phys Chem Lett. 2017 Apr 6;8(7):1489-1495. doi: 10.1021/acs.jpclett.7b00348. Epub 2017 Mar 20.
Because of growing applications in advanced fluorescence imaging, the mechanisms and dynamics of photoinduced reactions in reversibly photoswitchable fluorescent proteins are currently attracting much interest. We report the first time-resolved study of the photoswitching of Dreiklang, so far the only fluorescent protein to undergo reversible photoinduced chromophore hydration. Using broadband femtosecond transient absorption spectroscopy, we show that the reaction is triggered by an ultrafast deprotonation of the chromophore phenol group in the excited state in 100 fs. This primary step is accompanied by coherent oscillations that we assign to its coupling with a low-frequency mode, possibly a deformation of the chromophore hydrogen bond network. A ground-state intermediate is formed in the picosecond-nanosecond regime that we tentatively assign to the deprotonated water adduct. We suggest that proton ejection from the phenol group leads to a charge transfer from the phenol to the imidazolinone ring, which triggers imidazolinone protonation by nearby Glu222 and catalyzes the addition of the water molecule.
由于在先进荧光成像中的应用不断增加，可逆光开关荧光蛋白中光诱导反应的机制和动力学目前引起了广泛关注。我们首次对Dreiklang的光开关进行了时间分辨研究，Dreiklang是迄今为止唯一一种经历可逆光诱导发色团水合作用的荧光蛋白。使用宽带飞秒瞬态吸收光谱，我们表明该反应是由激发态下发色团酚基团在100飞秒内的超快去质子化引发的。这一初级步骤伴随着相干振荡，我们将其归因于与低频模式的耦合，可能是发色团氢键网络的变形。在皮秒-纳秒时间范围内形成了一个基态中间体，我们初步将其归因于去质子化的水加合物。我们认为，酚基团的质子喷射导致电荷从酚转移到咪唑啉酮环，这引发了附近Glu222对咪唑啉酮的质子化，并催化了水分子的加成。
