Chattoraj M, King B A, Bublitz G U, Boxer S G
Department of Chemistry, Stanford University, CA 94305-5080, USA.
Proc Natl Acad Sci U S A. 1996 Aug 6;93(16):8362-7. doi: 10.1073/pnas.93.16.8362.
The green fluorescent protein (GFP) 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 photo-excitation 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.
自发现维多利亚多管发光水母的绿色荧光蛋白（GFP）的发色团是由一个六肽单元的自催化、翻译后环化和氧化产生以来，它引起了广泛关注。这使得GFP的DNA序列能够与任何蛋白质的DNA序列融合，然后通过灵敏的荧光方法可以很容易地监测该蛋白质的表达或转运，而无需添加外源荧光染料。利用荧光上转换光谱法（时间分辨率约为100飞秒），在可见光区对GFP的两个强吸收带分别进行光激发后，研究了GFP的激发态动力学。结果表明，较高能量特征的激发会非常迅速地产生较低能量形式的物质，并且通过用氘取代可交换质子，激发态的相互转化率可以显著减慢。这一观察结果及其他结果引出了一个模型，其中两个可见吸收带对应于处于两种基态构象的GFP。这些构象在基态下可以缓慢相互转化，但在激发态下这个过程要快得多。观察到的同位素效应表明，初始激发态过程涉及一个质子转移反应，随后是额外的结构变化。这些观察结果可能有助于解释和推动那些改变吸收特性并提高GFP光稳定性的突变。
