Ultra-fast excited state dynamics in green fluorescent protein: Multiple states and proton transfer
Ultra-fast excited state dynamics in green fluorescent protein: Multiple states and proton transfer
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DOI：
10.1073/pnas.93.16.8362
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发表时间：
1996-08-06
影响因子：
11.1
通讯作者：
Boxer, SG
中科院分区：
文献类型：
--
作者：
Chattoraj, M;King, BA;Boxer, SG
自从发现水母的绿色荧光蛋白(GPP)是由六肽单元的自身催化、翻译后环化和氧化产生的发色团以来，引起了人们的广泛兴趣。这使得GFP的DNA序列可以与任何蛋白质的DNA序列融合，这些蛋白质的表达或运输可以很容易地通过敏感的荧光方法进行监测，而不需要添加外源荧光染料。利用荧光上转换光谱(时间分辨率约为100ps)研究了绿色荧光蛋白在可见光中的两个强吸收带光激发后的激发态动力学。结果表明，较高能量特征的激发很快就会导致一种较低能量组态的形式，而用可交换质子取代可交换质子可以显著地减慢激发态的相互转换速率。这一观察结果和其他人提出了一个模型，其中两个可见吸收带以两个基态构象对应于GFP。这些构象在基态下可以缓慢地相互转化，但在激发态时这一过程要快得多。观察到的同位素效应表明，初始激发态过程涉及质子转移反应，随后发生额外的结构变化。这些观察结果可能有助于合理化和激发改变吸收性质的突变，并提高GFP的光稳定性。
The green fluorescent protein (GPP) of the jellyfish Aequorea Victoria has attracted widespread interest since the discovery that its chromophore is generated by the autocatalytic, posttranslational cyclization and oxidation of a hexapeptide unit. This permits fusion of the DNA sequence of GFP with that of any protein whose expression or transport can then be readily monitored by sensitive fluorescence methods without the need to add exogenous fluorescent dyes. The excited state dynamics of GFP were studied following photoexcitation of each of its two strong absorption bands in the visible using fluorescence upconversion spectroscopy (about 100 fs time resolution). It is shown that excitation of the higher energy feature leads very rapidly to a form of the lower energy species, and that the excited state interconversion rate can be markedly slowed by replacing exchangeable protons with deuterons, This observation and others lead to a model in which the two visible absorption bands correspond to GFP in two ground-state conformations. These conformations can be slowly interconverted in the ground state, but the process is much faster in the excited state. The observed isotope effect suggests that the initial excited state process involves a proton transfer reaction that is followed by additional structural changes. These observations may help to rationalize and motivate mutations that alter the absorption properties and improve the photo stability of GFP.