Wachter R M, King B A, Heim R, Kallio K, Tsien R Y, Boxer S G, Remington S J
Institute of Molecular Biology, Department of Physics, University of Oregon, Eugene 97403, USA.
Biochemistry. 1997 Aug 12;36(32):9759-65. doi: 10.1021/bi970563w.
The crystal structure of a blue emission variant (Y66H/Y145F) of the Aequorea victoria green fluorescent protein has been determined by molecular replacement and the model refined. The crystallographic R-factor is 18.1% for all data from 20 to 2.1 A, and the model geometry is excellent. The chromophore is non-native and is autocatalytically generated from the internal tripeptide Ser65-His66-Gly67. The final electron density maps indicate that the formation of the chromophore is complete, including 1,2 dehydration of His66 as indicated by the planarity of the chromophore. The chromophore is in the cis conformation, with no evidence for any substantial fraction of the trans configuration or uncyclized apoprotein, and is well-shielded from bulk solvent by the folded protein. These characteristics indicate that the machinery for production of the chromophore from a buried tripeptide unit is not only intact but also highly efficient in spite of a major change in chromophore chemical structure. Nevertheless, there are significant rearrangements in the hydrogen bond configuration around the chromophore as compared to wild-type, indicating flexibility of the active site. pH titration of the intact protein and the chromopeptide (pKa1 = 4.9 +/- 0.1, pKa2 = 12.0 +/- 0.1) suggests that the predominant form of the chromophore in the intact protein is electrically neutral. In contrast to the wild-type protein [Chattoraj, M., King, B. A., Bublitz, G. U., & Boxer, S. G. (1996) Proc. Natl. Acad. Sci. U.S.A., 8362-8367], femtosecond fluorescence up-conversion spectroscopy of the intact protein and a partially deuterated form strongly suggests that excited-state proton transfer is not coupled to fluorescence emission.
通过分子置换法测定了维多利亚多管水母绿色荧光蛋白蓝色发射变体（Y66H/Y145F）的晶体结构，并对模型进行了优化。对于20至2.1 Å的所有数据，晶体学R因子为18.1%，模型几何结构良好。发色团是非天然的，由内部三肽Ser65-His66-Gly67自催化生成。最终的电子密度图表明发色团的形成是完整的，发色团的平面性表明His66发生了1,2脱水反应。发色团呈顺式构象，没有证据表明存在任何可观比例的反式构型或未环化的脱辅基蛋白，并且被折叠的蛋白质很好地与大量溶剂屏蔽开来。这些特征表明，尽管发色团化学结构发生了重大变化，但从埋藏的三肽单元产生发色团的机制不仅完整而且高效。然而，与野生型相比，发色团周围的氢键构型有显著重排，表明活性位点具有灵活性。完整蛋白质和发色肽的pH滴定（pKa1 = 4.9 ± 0.1，pKa2 = 12.0 ± 0.1）表明，完整蛋白质中发色团的主要形式是电中性的。与野生型蛋白质[Chattoraj, M., King, B. A., Bublitz, G. U., & Boxer, S. G. (1996) Proc. Natl. Acad. Sci. U.S.A., 8362-8367]不同，完整蛋白质和部分氘代形式的飞秒荧光上转换光谱强烈表明，激发态质子转移与荧光发射不耦合。
