Forschungszentrum Jülich, ICS-5, 52425 Jülich, Germany.
RWTH Aachen University, I. Physikalisches Institut (IA), 52056 Aachen, Germany.
Molecules. 2018 Nov 27;23(12):3105. doi: 10.3390/molecules23123105.
Bacterial periplasmic binding proteins (PBPs) undergo a pronounced ligand-induced conformational change which can be employed to monitor ligand concentrations. The most common strategy to take advantage of this conformational change for a biosensor design is to use a Förster resonance energy transfer (FRET) signal. This can be achieved by attaching either two fluorescent proteins (FPs) or two organic fluorescent dyes of different colors to the PBPs in order to obtain an optical readout signal which is closely related to the ligand concentration. In this study we compare a FP-equipped and a dye-labeled version of the glucose/galactose binding protein MglB at the single-molecule level. The comparison demonstrates that changes in the FRET signal upon glucose binding are more pronounced for the FP-equipped sensor construct as compared to the dye-labeled analog. Moreover, the FP-equipped sensor showed a strong increase of the FRET signal under crowding conditions whereas the dye-labeled sensor was not influenced by crowding. The choice of a labeling scheme should therefore be made depending on the application of a FRET-based sensor.
细菌周质结合蛋白 (PBPs) 经历明显的配体诱导构象变化，可用于监测配体浓度。最常见的利用这种构象变化进行生物传感器设计的策略是使用Förster 共振能量转移 (FRET) 信号。这可以通过将两个荧光蛋白 (FPs) 或两种不同颜色的有机荧光染料附着到 PBPs 上来实现，以获得与配体浓度密切相关的光学读出信号。在这项研究中，我们在单分子水平上比较了葡萄糖/半乳糖结合蛋白 MglB 的 FP 装备和染料标记版本。比较表明，与染料标记的类似物相比，葡萄糖结合后 FRET 信号的变化对于 FP 装备的传感器构建更为明显。此外，FP 装备的传感器在拥挤条件下显示出 FRET 信号的强烈增加，而染料标记的传感器不受拥挤的影响。因此，应根据 FRET 基传感器的应用选择标记方案。
