Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan.
Sci Rep. 2024 Oct 1;14(1):22832. doi: 10.1038/s41598-024-73696-y.
Green fluorescent protein (GFP) is widely utilized as a fluorescent tag in biochemical fields. Whereas the intermediate (I) state has been proposed in the photoreaction cycle in addition to the A and B states, until now the structure of I has only been estimated by computational studies. In this paper, we report the crystal structures of the I stabilizing variants of GFP at high resolutions where respective atoms can be observed separately. Comparison with the structures in the other states highlights the structural feature of the I state. The side chain of one of the substituted residues, Val203, adopts the gauche- conformation observed for Thr203 in the A state, which is different from the B state. On the other hand, His148 interacts with the chromophore by ordinary hydrogen bonding with a distance of 2.85 Å, while the weaker interaction by longer distances is observed in the A state. Therefore, it was indicated that it is possible to distinguish three states A, B and I by the two hydrogen bond distances Oγ-Thr203···Oη-chromophore and Nδ1-His148···Oη-chromophore. We discuss the characteristics of the I intermediate of wild-type GFP on the bases of the structure estimated from the variant structures by quantum chemical calculations.
绿色荧光蛋白（GFP）广泛用作生化领域的荧光标记物。尽管在光反应循环中除了 A 和 B 态之外还提出了中间（I）态，但直到现在，I 态的结构仅通过计算研究来估计。在本文中，我们报告了 GFP 的 I 稳定变体在高分辨率下的晶体结构，在该分辨率下可以分别观察到各个原子。与其他状态下的结构进行比较突出了 I 态的结构特征。取代残基之一 Val203 的侧链采用了在 A 态中观察到的 Thr203 的 gauche-构象，这与 B 态不同。另一方面，His148 通过 2.85Å 的普通氢键与发色团相互作用，而在 A 态中观察到较弱的通过较长距离的相互作用。因此，表明可以通过两个氢键距离 Oγ-Thr203···Oη-chromophore 和 Nδ1-His148···Oη-chromophore 来区分 A、B 和 I 三种状态。我们根据变体结构通过量子化学计算估计的结构讨论了野生型 GFP 的 I 中间态的特征。
