Cardoso Ramos Felipe, Cupellini Lorenzo, Mennucci Benedetta
Dipartimento di Chimica e Chimica Industriale, University of Pisa, Via G. Moruzzi 13, Pisa, I-56124, Italy.
J Phys Chem B. 2021 Feb 25;125(7):1768-1777. doi: 10.1021/acs.jpcb.0c10834. Epub 2021 Feb 10.
Flavin-based fluorescent proteins are a class of fluorescent reporters derived from light, oxygen, and voltage (LOV) sensing proteins. Through mutagenesis, natural LOV proteins have been engineered to obtain improved fluorescence properties. In this study, we combined extended classical Molecular Dynamics simulations and multiscale Quantum Mechanics/Molecular Mechanics methods to clarify the relationship between structural and dynamic changes induced by specific mutations and the spectroscopic response. To reach this goal we compared two LOV variants, one obtained by the single mutation needed to photochemically inactivate the natural system, and the other (iLOV) obtained through additional mutations and characterized by a significantly improved fluorescence. Our simulations confirmed the "flipping and crowding" effect induced in iLOV by the additional mutations and revealed its mechanism of action. We also showed that these mutations, and the resulting differences in the composition and flexibility of the binding pockets, are not reflected in significant shifts of the excitation and emission energies, in agreement with the similarity of the spectra measured for the two systems. However, a small but consistent reduction was found in the Stokes shift of iLOV, suggesting a reduction of the intermolecular reorganization experienced by the chromophore after excitation, which could slow down its internal conversion to the ground state and improve the fluorescence.
黄素基荧光蛋白是一类源自光、氧和电压（LOV）传感蛋白的荧光报告分子。通过诱变，对天然LOV蛋白进行了改造，以获得更好的荧光特性。在本研究中，我们结合了扩展的经典分子动力学模拟和多尺度量子力学/分子力学方法，以阐明特定突变引起的结构和动态变化与光谱响应之间的关系。为实现这一目标，我们比较了两种LOV变体，一种是通过光化学灭活天然系统所需的单突变获得的，另一种（iLOV）是通过额外突变获得的，其荧光显著改善。我们的模拟证实了额外突变在iLOV中诱导的“翻转和拥挤”效应，并揭示了其作用机制。我们还表明，这些突变以及由此导致的结合口袋组成和灵活性的差异，并未反映在激发和发射能量的显著变化中，这与两个系统测量光谱的相似性一致。然而，发现iLOV的斯托克斯位移有一个小但一致的减小，这表明激发后发色团经历的分子间重组减少，这可能会减缓其向基态的内部转换并改善荧光。
