Drobizhev Mikhail, Molina Rosana S, Callis Patrik R, Scott J Nathan, Lambert Gerard G, Salih Anya, Shaner Nathan C, Hughes Thomas E
Department of Cell Biology and Neuroscience, Montana State University, Bozeman, MT, United States.
Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT, United States.
Front Mol Biosci. 2021 Feb 3;8:633217. doi: 10.3389/fmolb.2021.633217. eCollection 2021.
Genetically encoded probes with red-shifted absorption and fluorescence are highly desirable for imaging applications because they can report from deeper tissue layers with lower background and because they provide additional colors for multicolor imaging. Unfortunately, red and especially far-red fluorescent proteins have very low quantum yields, which undermines their other advantages. Elucidating the mechanism of nonradiative relaxation in red fluorescent proteins (RFPs) could help developing ones with higher quantum yields. Here we consider two possible mechanisms of fast nonradiative relaxation of electronic excitation in RFPs. The first, known as the energy gap law, predicts a steep exponential drop of fluorescence quantum yield with a systematic red shift of fluorescence frequency. In this case the relaxation of excitation occurs in the chromophore without any significant changes of its geometry. The second mechanism is related to a twisted intramolecular charge transfer in the excited state, followed by an ultrafast internal conversion. The chromophore twisting can strongly depend on the local electric field because the field can affect the activation energy. We present a spectroscopic method of evaluating local electric fields experienced by the chromophore in the protein environment. The method is based on linear and two-photon absorption spectroscopy, as well as on quantum-mechanically calculated parameters of the isolated chromophore. Using this method, which is substantiated by our molecular dynamics simulations, we obtain the components of electric field in the chromophore plane for seven different RFPs with the same chromophore structure. We find that in five of these RFPs, the nonradiative relaxation rate increases with the strength of the field along the chromophore axis directed from the center of imidazolinone ring to the center of phenolate ring. Furthermore, this rate depends on the corresponding electrostatic energy change (calculated from the known fields and charge displacements), in quantitative agreement with the Marcus theory of charge transfer. This result supports the dominant role of the twisted intramolecular charge transfer mechanism over the energy gap law for most of the studied RFPs. It provides important guidelines of how to shift the absorption wavelength of an RFP to the red, while keeping its brightness reasonably high.
具有红移吸收和荧光特性的基因编码探针对于成像应用非常理想，因为它们可以从更深的组织层进行报告，背景较低，并且为多色成像提供了额外的颜色。不幸的是，红色尤其是远红荧光蛋白的量子产率非常低，这削弱了它们的其他优势。阐明红色荧光蛋白（RFP）中非辐射弛豫的机制有助于开发具有更高量子产率的蛋白。在这里，我们考虑RFP中电子激发快速非辐射弛豫的两种可能机制。第一种，称为能隙定律，预测随着荧光频率的系统性红移，荧光量子产率会急剧指数下降。在这种情况下，激发的弛豫发生在发色团中，其几何结构没有任何显著变化。第二种机制与激发态下的扭曲分子内电荷转移有关，随后是超快的内转换。发色团的扭曲可能强烈依赖于局部电场，因为电场会影响活化能。我们提出了一种光谱方法来评估蛋白质环境中发色团所经历的局部电场。该方法基于线性和双光子吸收光谱，以及分离发色团的量子力学计算参数。使用这种由我们的分子动力学模拟证实的方法，我们获得了七种具有相同发色团结构的不同RFP在发色团平面内的电场分量。我们发现，在其中五种RFP中，非辐射弛豫速率随着沿从咪唑啉酮环中心指向酚盐环中心的发色团轴的电场强度增加而增加。此外，该速率取决于相应的静电能变化（根据已知电场和电荷位移计算），与电荷转移的马库斯理论在定量上一致。这一结果支持了在大多数研究的RFP中，扭曲分子内电荷转移机制比能隙定律起主导作用。它为如何将RFP的吸收波长红移，同时保持其亮度相当高提供了重要指导。
