Inst. of Organic Chemistry and Biochemistry CAS, Flemingovo nám. 2, 160 00, Prague 6, Czech Republic.
1st Faculty of Medicine, Charles University, Albertov 4, 128 00, Prague 2, Czech Republic.
Phys Chem Chem Phys. 2023 Aug 23;25(33):22117-22123. doi: 10.1039/d3cp01242e.
Molecules of fluorescent proteins (FPs) exhibit distinct optical directionality. This optical directionality is characterized by transition dipole moments (TDMs), and their orientation with respect to the molecular structures. Although our recent observations of FP crystals allowed us to determine the mean TDM directions with respect to the framework of representative FP molecules, the dynamics of TDM orientations within FP molecules remain to be ascertained. Here we describe the results of our investigations of the dynamics of TDM directions in the fluorescent proteins eGFP, mTurquoise2 and mCherry, through time-resolved fluorescence polarization measurements and microsecond time scale all-atom molecular dynamics (MD) simulations. The investigated FPs exhibit initial fluorescence anisotropies () consistent with significant differences in the orientation of the excitation and emission TDMs. However, based on MD data, we largely attribute this observation to rapid (sub-nanosecond) fluorophore motions within the FP molecular framework. Our results allow improved determinations of orientational distributions of FP molecules by polarization microscopy, as well as more accurate interpretations of fluorescence resonance energy transfer (FRET) observations.
荧光蛋白（FP）的分子表现出明显的光各向异性。这种光各向异性的特点是跃迁偶极矩（TDM）及其相对于分子结构的方向。尽管我们最近对 FP 晶体的观察使我们能够确定相对于代表性 FP 分子框架的平均 TDM 方向，但 FP 分子内 TDM 方向的动力学仍有待确定。在这里，我们通过时间分辨荧光偏振测量和微秒时间尺度全原子分子动力学（MD）模拟，描述了我们对荧光蛋白 eGFP、mTurquoise2 和 mCherry 中 TDM 方向动力学的研究结果。所研究的 FP 表现出与激发和发射 TDM 方向显著差异一致的初始荧光各向异性（）。然而，根据 MD 数据，我们主要将这一观察归因于 FP 分子框架内荧光团的快速（亚纳秒）运动。我们的结果允许通过偏振显微镜更准确地确定 FP 分子的取向分布，并更准确地解释荧光共振能量转移（FRET）观察结果。
