Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto-Daigaku-Katsura, Nshikyo-ku, Kyoto 615-8510, Japan.
J Phys Chem B. 2010 Mar 4;114(8):2971-9. doi: 10.1021/jp9099573.
The emitting states of green fluorescent protein (GFP), monomeric Kusabira orange (mKO), and Discosoma red (DsRed) were studied using QM/MM and SAC-CI methods. By comparing the electronic structures among the green-, orange-, and red-emitting states as well as their electrostatic and quantum mechanical interactions within the protein cavity, the basic mechanisms for determining emission colors have been clarified. We found that the orange and red emissions of mKO and DsRed, respectively, result from cancellation between two effects, the pi skeleton extension (red shift) and protein electrostatic potential (blue shift). The extension of the pi skeleton enhances the intramolecular charge-transfer character of the transition, which makes the fluorescence energy more sensitive to the protein's electrostatic potential. On the basis of this mechanism, we predicted amino acid mutations that could red shift the emission energy of DsRed. A novel single amino acid mutation, which was examined computationally, reduced the DsRed emission energy from 2.14 (579 nm) to 1.95 eV (636 nm), which is approaching near-infrared fluorescence.
采用量子力学/分子力学（QM/MM）和激发态组态相互作用（SAC-CI）方法研究了绿色荧光蛋白（GFP）、单体 Kusabira 橙（mKO）和 Discosoma 红（DsRed）的发光状态。通过比较绿、橙、红发射态之间的电子结构以及它们在蛋白腔体内的静电和量子力学相互作用，阐明了决定发射颜色的基本机制。我们发现，mKO 和 DsRed 的橙色和红色发射分别来自两个效应的抵消，即π骨架延伸（红移）和蛋白质静电势（蓝移）。π骨架的延伸增强了跃迁的分子内电荷转移特性，使荧光能量对蛋白质的静电势更加敏感。基于这一机制，我们预测了可以使 DsRed 发射能量红移的氨基酸突变。一种新的单氨基酸突变，经计算检验，将 DsRed 的发射能量从 2.14 eV（579 nm）降低到 1.95 eV（636 nm），接近近红外荧光。
