酶结合 NADH 中的双光子激发荧光动力学：荧光衰减时间和各向异性弛豫的异质性,The Journal of Physical Chemistry B

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酶结合 NADH 中的双光子激发荧光动力学：荧光衰减时间和各向异性弛豫的异质性
The Journal of Physical Chemistry B ( IF 2.8 ) Pub Date : 2021-08-19 , DOI: 10.1021/acs.jpcb.1c04226
Ioanna A Gorbunova ₁ , Maxim E Sasin ₁ , Dmitrii P Golyshev _{1,

2} , Alexander A Semenov ₁ , Andrey G Smolin ₁ , Yaroslav M Beltukov ₁ , Oleg S Vasyutinskii ₁

Affiliation

已使用 TCSPC 光谱研究了缓冲溶液中乙醇脱氢酶 (ADH) 中 NADH 的偏振荧光动力学。使用全局拟合程序来确定来自实验的荧光参数。所得结果的解释得到了ab initio 的支持NADH结构的计算。开发了一个理论模型来描述 ADH-NADH 复合物中的偏振荧光衰减，该模型考虑了几种相互作用场景。使用多指数拟合模型对偏振不敏感荧光衰减进行了比较分析。如图所示，ADH-NADH 复合物中衰减时间显着增加的起源可归因于在非极性结合位点环境条件下烟酰胺环中非辐射弛豫率的降低。与在游离 NADH 中观察到的两个衰变时间相比，ADH-NADH 复合物中单个衰变时间的存在归因于 ADH 结合位点中的单个 NADH 未折叠构象。

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Two-Photon Excited Fluorescence Dynamics in Enzyme-Bound NADH: the Heterogeneity of Fluorescence Decay Times and Anisotropic Relaxation

The dynamics of polarized fluorescence in NADH in alcohol dehydrogenase (ADH) in buffer solution has been studied using the TCSPC spectroscopy. A global fit procedure was used for determination of the fluorescence parameters from experiment. The interpretation of the results obtained was supported by ab initio calculations of the NADH structure. A theoretical model was developed describing the polarized fluorescence decay in ADH–NADH complexes that considered several interaction scenarios. A comparative analysis of the polarization-insensitive fluorescence decay using multiexponential fitting models has been carried out. As shown, the origin of a significant enhancement of the decay time in the ADH–NADH complex can be attributed to the decrease of nonradiative relaxation rates in the nicotinamide ring in the conditions of the apolar binding site environment. The existence of a single decay time in the ADH–NADH complex in comparison with two decay times observed in free NADH was attributed to a single NADH unfolded conformation in the ADH binding site. Comparison of the experimental data with the theoretical model suggested the existence of an anisotropic relaxation time of about 1 ns that is related with the rotation of fluorescence transition dipole moment due to the rearrangement of the excited state NADH nuclear configuration.

更新日期：2021-09-02

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