Ground-state proton transfer kinetics in green fluorescent protein.
Ground-state proton transfer kinetics in green fluorescent protein.
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DOI：
10.1021/bi500147n
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发表时间：
2014-09-23
期刊：
影响因子：
2.9
通讯作者：
Boxer SG
中科院分区：
文献类型：
--
作者：
Oltrogge LM;Wang Q;Boxer SG
质子转移在绿色荧光蛋白（GFP）的光学性质中起着重要作用。虽然人们对GFP中发生在超快时间尺度上的激发态质子转移反应（ESPT）了解甚多，但对控制基态质子转移速率和途径的因素了解相对较少。我们利用一种特定的同位素标记策略，结合一维13C核磁共振（NMR）光谱，安装和监测直接靠近GFP发色团电离位点的13C。该探针的化学位移对发色团的质子化状态高度敏感，所得光谱反映了核磁共振谱线形状中质子转移的热力学和动力学。该信息由时间分辨核磁共振，荧光相关光谱，稳态吸光度和荧光测量补充，以提供一个画面的发色团电离反应跨越广泛的时间域。我们的研究结果表明，GFP中的质子转移可以用两个位点模型很好地描述，在这个模型中，发色团与一个次要位点（可能是ESPT的末端质子受体Glu222）能量耦合。此外，选择绿色荧光蛋白圆形置换体的实验表明，第七股β-链的结构动力学在控制质子从体溶液转移到埋藏的发色团中起着重要作用。
Proton transfer plays an important role in the optical properties of green fluorescent protein (GFP). While much is known about excited-state proton transfer reactions (ESPT) in GFP occurring on ultrafast time scales, comparatively little is understood about the factors governing the rates and pathways of ground-state proton transfer. We have utilized a specific isotopic labeling strategy in combination with one-dimensional 13C nuclear magnetic resonance (NMR) spectroscopy to install and monitor a 13C directly adjacent to the GFP chromophore ionization site. The chemical shift of this probe is highly sensitive to the protonation state of the chromophore, and the resulting spectra reflect the thermodynamics and kinetics of the proton transfer in the NMR line shapes. This information is complemented by time-resolved NMR, fluorescence correlation spectroscopy, and steady-state absorbance and fluorescence measurements to provide a picture of chromophore ionization reactions spanning a wide time domain. Our findings indicate that proton transfer in GFP is described well by a two-site model in which the chromophore is energetically coupled to a secondary site, likely the terminal proton acceptor of ESPT, Glu222. Additionally, experiments on a selection of GFP circular permutants suggest an important role played by the structural dynamics of the seventh β-strand in gating proton transfer from bulk solution to the buried chromophore.
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