Department of Chemistry, Lomonosov Moscow State University, Leninskie Gory 1/3, 119991 Moscow, Russia.
Int J Mol Sci. 2023 Jul 10;24(14):11266. doi: 10.3390/ijms241411266.
The search for fluorescent proteins with large two-photon absorption (TPA) cross-sections and improved brightness is required for their efficient use in bioimaging. Here, we explored the impact of a single-point mutation close to the anionic form of the GFP chromophore on its TPA activity. We considered the lowest-energy transition of EGFP and its modification EGFP T203I. We focused on a methodology for obtaining reliable TPA cross-sections for mutated proteins, based on conformational sampling using molecular dynamics simulations and a high-level XMCQDPT2-based QM/MM approach. We also studied the numerical convergence of the sum-over-states formalism and provide direct evidence for the applicability of the two-level model for calculating TPA cross-sections in EGFP. The calculated values were found to be very sensitive to changes in the permanent dipole moments between the ground and excited states and highly tunable by internal electric field of the protein environment. In the case of the GFP chromophore anion, even a single hydrogen bond was shown to be capable of drastically increasing the TPA cross-section. Such high tunability of the nonlinear photophysical properties of the chromophore anions can be used for the rational design of brighter fluorescent proteins for bioimaging using two-photon laser scanning microscopy.
寻找具有大双光子吸收（TPA）截面和改进亮度的荧光蛋白，对于它们在生物成像中的有效应用是必需的。在这里，我们研究了靠近 GFP 发色团阴离子形式的单点突变对其 TPA 活性的影响。我们考虑了 EGFP 的最低能量跃迁及其修饰 EGFP T203I。我们专注于一种基于分子动力学模拟和基于高级 XMCQDPT2 的QM/MM 方法进行构象采样的方法，以获得突变蛋白可靠的 TPA 截面。我们还研究了态和求和公式的数值收敛性，并为在 EGFP 中计算 TPA 截面的两能级模型的适用性提供了直接证据。计算值发现对基态和激发态之间的永久偶极矩的变化非常敏感，并且可以通过蛋白质环境的内电场高度调节。在 GFP 发色团阴离子的情况下，甚至单个氢键就被证明能够显著增加 TPA 截面。这种发色团阴离子的非线性光物理性质的高可调节性可用于使用双光子激光扫描显微镜进行生物成像的更亮荧光蛋白的合理设计。
