Effects of threonine 203 replacements on excited-state dynamics and fluorescence properties of the green fluorescent protein (GFP)
Effects of threonine 203 replacements on excited-state dynamics and fluorescence properties of the green fluorescent protein (GFP)
复制标题
DOI：
10.1021/jp9942522
复制
发表时间：
2000-05-18
影响因子：
3.3
通讯作者：
Michel-Beyerle, ME
中科院分区：
文献类型：
--
作者：
Kummer, AD;Wiehler, J;Michel-Beyerle, ME
我们报告了野生型绿色荧光蛋白（GFP）和单位点突变体的比较研究，其中203位的苏氨酸已被脂肪族和芳香族残基取代，即缬氨酸（V）、异亮氨酸（I）、苯丙氨酸（F）、酪氨酸（Y）和组氨酸（H）。稳态吸收光谱揭示了与野生型 GFP 相比反映突变体不同电荷分布的变化。虽然质子化荧光团 RH 的吸收峰在所有 T203 突变体中仅经历很小的红移，但在去质子化形式 R- 中观察到明显的红移，大约 100%。脂肪族突变体 T203V 和 T203I 约 1000 cm(-1)。 T203F 为 1200 厘米(-1)，T203Y 为 1360 厘米(-1)。因此，我们得出结论，能量高于野生型 R 态的基态构象是所有研究的 T203 突变体中红移的主要根源。此外，RH 和 R- 基态平衡的突变依赖性变化至少由两种静电稳定模式引起，一种基于氢键，如 T203，另一种基于 pi-pi 堆积，如 T203F 和 T203Y。令人惊讶的是，RH* 的去质子化动力学仅受 203 位突变的微弱影响。仅在红移最大的突变体 T203Y 中观察到了 RH* 的额外超快 (1.7 ps) 激发态衰变通道。 T203Y 中 RH* 的衰变和 RH 基态恢复这两个过程的相同动力学，根据两种机制进行了讨论：(i) 决定速率的电子从质子化（或去质子化）酪氨酰 203 残基转移到 RH*，随后是相当快的重组过程，由于能量原因，这在 T203F 中不会发生；(ii) 围绕外环双键的旋转运动有利于 RH* 的内部转换。
We report a comparative study of wild-type green fluorescent protein (GFP) and single-site mutants in which threonine at position 203 has been replaced by aliphatic and aromatic residues, i.e., by valine (V), isoleucine (I), phenylalanine (F), tyrosine (Y), and histidine (H). Steady-state absorption spectra reveal changes that reflect different charge distributions in the mutants as compared to wild-type GFP. While the absorption peak of the protonated fluorophore, RH, undergoes only a small red shift in all T203 mutants, a pronounced red shift is observed for the deprotonated form R-, ca. 1000 cm(-1) for the aliphatic mutants T203V and T203I, ca. 1200 cm(-1) for T203F, and 1360 cm(-1) for T203Y. Thus, we conclude that a ground-state conformation higher in energy than the wild-type R- state is the predominant origin of the red shift in all the T203 mutants investigated. Furthermore, mutant-dependent changes in the ground-state equilibria of RH and R- result from at least two modes of electrostatic stabilization, one resting on hydrogen bonding as in T203 and the other one on pi-pi-stacking as in T203F and T203Y. Surprisingly, the deprotonation dynamics of RH* is only weakly affected by the mutations at position 203. Only in the most red-shifted mutant T203Y an additional ultrafast (1.7 ps) excited-state decay channel of RH* has been observed. The identical kinetics of both processes, decay of RH* and ground-state recovery of RH in T203Y, is discussed in terms of two mechanisms: (i) rate-determining electron transfer from the protonated (or deprotonated) tyrosyl 203 residue to RH* followed by considerably faster recombination processes, which cannot occur in T203F for energetic reasons, and (ii) internal conversion in RH* favored by rotational motion around the exocyclic double bond.