Department of Chemistry, Imperial College London, Wood Lane, London, W12 0BZ, United Kingdom.
Methods Appl Fluoresc. 2019 Oct 10;7(4):044004. doi: 10.1088/2050-6120/ab481f.
Viscosity sensitive fluorophores termed 'molecular rotors' represent a convenient and quantitative tool for measuring intracellular viscosity via Fluorescence Lifetime Imaging Microscopy (FLIM). We compare the FLIM performance of two BODIPY-based molecular rotors bound to HaloTag protein expressed in different subcellular locations. While both rotors are able to penetrate live cells and specifically label the desired intracellular location, we found that the rotor with a longer HaloTag protein recognition motif was significantly affected by photo-induced damage when bound to the HaloTag protein, while the other dye showed no changes upon irradiation. Molecular dynamics modelling indicates that the irradiation-induced electron transfer between the BODIPY moiety and the HaloTag protein is a plausible explanation for these photostability issues. Our results demonstrate that binding to the targeted protein may significantly alter the photophysical behaviour of a fluorescent probe and therefore its thorough characterisation in the protein bound form is essential prior to any in vitro and in cellulo applications.
黏度敏感荧光团被称为“分子转子”，是通过荧光寿命成像显微镜（FLIM）测量细胞内黏度的一种便捷且定量的工具。我们比较了两种结合在不同亚细胞位置表达的 HaloTag 蛋白上的基于 BODIPY 的分子转子的 FLIM 性能。虽然这两种转子都能够穿透活细胞并特异性标记所需的细胞内位置，但我们发现，当与 HaloTag 蛋白结合时，具有较长 HaloTag 蛋白识别基序的转子受到光诱导损伤的显著影响，而另一种染料在照射时没有变化。分子动力学模拟表明，BODIPY 部分和 HaloTag 蛋白之间的光诱导电子转移是造成这些光稳定性问题的一个合理解释。我们的结果表明，与靶蛋白的结合可能会显著改变荧光探针的光物理行为，因此在进行任何体外和细胞内应用之前，对其在结合蛋白形式下的全面特性进行彻底表征是至关重要的。
