Leverhulme Quantum Biology Doctoral Training Centre, University of Surrey, Guildford GU2 7XH, UK.
Department of Microbial Sciences, School of Biosciences, University of Surrey, Guilford GU2 7XH, UK.
Phys Chem Chem Phys. 2023 Jul 26;25(29):19532-19539. doi: 10.1039/d3cp01765f.
Advances in ultra-fast photonics have enabled monitoring of biochemical interactions on a sub nano-second time scale. In addition, picosecond dynamics of intermolecular energy transfer in fluorescent proteins has been observed. Here, we present the development of a genetically encoded fluorescent sensor that can detect changes in hydrophobicity by monitoring ultrafast fluorescence depolarisation. Our sensor is composed of a pair of dimeric enhanced green fluorescent proteins (dEGFPs) linked by a flexible amino-acid linker. We show dimerisation is perturbed by the addition of glycerol which interferes with the hydrophobic interaction of the two proteins. Time-resolved fluorescence anisotropy revealed a systematic attenuation of ultrafast fluorescence depolarisation when the sensor was exposed to increasing glycerol concentrations. This suggests that as hydrophobicity increases, dEGFP pairing decreases within a tandem dimer. Un-pairing of the protein fluorophores dramatically alters the rate of energy transfer between the proteins, resulting in an increase in the limiting anisotropy of the sensor.
超快光子学的进步使得能够在亚纳秒时间尺度上监测生化相互作用。此外，还观察到了荧光蛋白中分子间能量转移的皮秒动力学。在这里，我们提出了一种遗传编码的荧光传感器的开发，该传感器可以通过监测超快荧光去偏振来检测疏水性的变化。我们的传感器由一对通过柔性氨基酸接头连接的二聚增强型绿色荧光蛋白（dEGFP）组成。我们表明，添加甘油会干扰两个蛋白质的疏水相互作用，从而破坏二聚化。时间分辨荧光各向异性表明，当传感器暴露于逐渐增加的甘油浓度时，超快荧光去偏振的系统衰减。这表明，随着疏水性的增加，串联二聚体中 dEGFP 配对减少。蛋白质荧光团的解偶联会极大地改变蛋白质之间能量转移的速率，从而导致传感器的极限各向异性增加。
