Local Electric Field Controls Fluorescence Quantum Yield of Red and Far-Red Fluorescent Proteins.
Local Electric Field Controls Fluorescence Quantum Yield of Red and Far-Red Fluorescent Proteins.
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DOI：
10.3389/fmolb.2021.633217
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发表时间：
2021
影响因子：
5
通讯作者：
Hughes TE
中科院分区：
文献类型：
--
作者：
Drobizhev M;Molina RS;Callis PR;Scott JN;Lambert GG;Salih A;Shaner NC;Hughes TE
具有红移吸收和荧光的基因编码探针对于成像应用来说是非常理想的，因为它们可以从具有较低背景的更深组织层进行报告，并且因为它们为多色成像提供额外的颜色。不幸的是，红色荧光蛋白，尤其是远红色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）的非辐射弛豫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。第一个定律称为能隙定律，它预测荧光量子产率会随着荧光频率的系统红移而急剧指数下降。在这种情况下，发色团中发生激发弛豫，而其几何形状没有任何显着变化。第二种机制与激发态下扭曲的分子内电荷转移以及随后的超快内部转换有关。发色团扭曲很大程度上取决于局部电场，因为电场会影响活化能。我们提出了一种评估蛋白质环境中发色团所经历的局部电场的光谱方法。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。使用这种经过我们的分子动力学模拟证实的方法，我们获得了具有相同发色团结构的七个不同 RFP 的发色团平面中的电场分量。我们发现，在其中五个 RFP 中，非辐射弛豫率随着沿着从咪唑啉酮环中心到酚盐环中心的发色团轴的场强而增加。此外，该速率取决于相应的静电能量变化（根据已知场和电荷位移计算），与马库斯电荷转移理论定量一致。这一结果支持了大多数研究的 RFP 中扭曲分子内电荷转移机制相对于能隙定律的主导作用。它为如何将 RFP 的吸收波长转移到红色，同时保持其亮度相当高提供了重要指导。
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.
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影响因子：
48
作者：
Gibson, Daniel G.;Young, Lei;Smith, Hamilton O.
通讯作者：
Smith, Hamilton O.
影响因子：
0.8
作者：
Drobizhev, Mikhail;Molina, Rosana S.;Hughes, Thomas E.
通讯作者：
Hughes, Thomas E.
影响因子：
3.3
作者：
Drobizhev, M.;Makarov, N. S.;Tillo, S. E.;Hughes, T. E.;Rebane, A.
通讯作者：
Rebane, A.
影响因子：
3.3
作者：
Ansbacher, Tamar;Srivastava, Hemant Kumar;Shurki, Avital
通讯作者：
Shurki, Avital
影响因子：
48
作者：
Bindels, Daphne S.;Haarbosch, Lindsay;Gadella, Theodorus W. J., Jr.
通讯作者：
Gadella, Theodorus W. J., Jr.