Kinetics of acid-induced spectral changes in the GFPmut2 chromophore
Kinetics of acid-induced spectral changes in the GFPmut2 chromophore
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DOI：
10.1021/ja045400r
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发表时间：
2005-01-19
影响因子：
15
通讯作者：
Mozzarelli, A
中科院分区：
文献类型：
--
作者：
Abbruzzetti, S;Grandi, E;Mozzarelli, A
我们已经使用了纳秒pH值跳跃技术，加上同时瞬态吸收和荧光发射检测，来表征GFPmut2发色团中酸诱导的光谱变化的动力学。在488 nm处的吸光度和绿色荧光发射的消失伴随着热激活的双指数弛豫发生。为了理解这两种瞬变的来源，我们在与发色团相互作用的氨基酸残基（H148G、T203V和E222Q）中引入了突变。结果表明，更快的瞬态与质子结合的解决方案，而第二个过程，较小的振幅，是由于周围的生色团的氨基酸的结构重排。质子化速率显示H148G突变体的3倍增加，表明His148在保护发色团免受溶剂影响方面起关键作用。T203V的去质子化速率小一个数量级，表明与Thr203的羟基的氢键在稳定去质子化形式的发色团中是重要的。一个动力学模型表明，除了保护发色团从溶剂中，His148可以作为主要受体的质子的方式发色团。
We have used a nanosecond pH-jump technique, coupled with simultaneous transient absorption and fluorescence emission detection, to characterize the dynamics of the acid-induced spectral changes in the GFPmut2 chromophore. Disappearance of the absorbance at 488 nm and the green fluorescence emission occurs with a thermally activated, double exponential relaxation. To understand the source of the two transients we have introduced mutations in amino acid residues that interact with the chromophore (H148G, T203V, and E222Q). Results indicate that the faster transient is associated with proton binding from the solution, while the second process, smaller in amplitude, is attributed to structural rearrangement of the amino acids surrounding the chromophore. The protonation rate shows a 3-fold increase for the H148G mutant, demonstrating that His148 plays a key role in protecting the chromophore from the solvent. The deprotonation rate for T203V is an order of magnitude smaller, showing that the hydrogen bond with the hydroxyl of Thr203 is important in stabilizing the deprotonated form of the chromophore. A kinetic model suggests that, in addition to protecting the chromophore from the solvent, His148 may act as the primary acceptor for the protons on the way to the chromophore.