Bizzarri Ranieri, Nifosì Riccardo, Abbruzzetti Stefania, Rocchia Walter, Guidi Sara, Arosio Daniele, Garau Gianpiero, Campanini Barbara, Grandi Elena, Ricci Fernanda, Viappiani Cristiano, Beltram Fabio
Scuola Normale Superiore-IIT Research Unit, Piazza dei Cavalieri 7 I-56126 Pisa, Italy. r.bizzarri@ sns.it
Biochemistry. 2007 May 8;46(18):5494-504. doi: 10.1021/bi602646r. Epub 2007 Apr 17.
The photophysical properties of most green fluorescent protein mutants (GFPs) are strongly affected by pH. This effect must be carefully taken into account when using GFPs as fluorescent probes or indicators. Usually, the pH-dependence of GFPs is rationalized on the basis of the ionization equilibrium of the chromophore phenol group. Yet many different mutants show spectral behavior that cannot be explained by ionization of this group alone. In this study, we propose a general model of protonation comprising two ionization sites (2S model). Steady-state optical measurements at different pH and temperature and pH-jump relaxation experiments were combined to highlight the thermodynamic and kinetic properties of paradigmatically different GFP variants. Our experiments support the 2S model. For the case of mutants in which E222 is the second protonation site, thermodynamic coupling between this residue's and the chromophore's ionization reactions was demonstrated. In agreement with the 2S model predictions, X-ray analysis of one of these mutants showed the presence of two chromophore populations at high pH.
大多数绿色荧光蛋白突变体（GFP）的光物理性质受pH值的强烈影响。在将GFP用作荧光探针或指示剂时，必须仔细考虑这种影响。通常，GFP的pH依赖性是基于发色团酚基团的电离平衡来解释的。然而，许多不同的突变体表现出的光谱行为不能仅通过该基团的电离来解释。在本研究中，我们提出了一个包含两个电离位点的质子化通用模型（2S模型）。结合在不同pH和温度下的稳态光学测量以及pH跃变弛豫实验，以突出典型不同的GFP变体的热力学和动力学性质。我们的实验支持2S模型。对于以E222作为第二个质子化位点的突变体，证明了该残基与发色团电离反应之间的热力学耦合。与2S模型预测一致，对其中一个突变体的X射线分析表明在高pH下存在两种发色团群体。
