Laboratory of Biophysics, Wageningen University & Research, Wageningen, The Netherlands.
Methods Mol Biol. 2024;2694:267-291. doi: 10.1007/978-1-0716-3377-9_13.
Single-molecule Förster resonance energy transfer (smFRET) is a powerful technique for the detection of conformational dynamics of biomolecules. While many smFRET experiments are performed using dye-labeled DNA, here we describe a comprehensive protocol to resolve the conformational dynamics of a protein system - notably from plasmid to data. Using the example of the heat-shock protein Hsp90, we describe the protein production and threefold site-specific bioconjugation, the smFRET measurement using total internal reflection fluorescence microscopy (TIRFM), and raw data processing to reveal time-resolved protein dynamics. The described smFRET approach is readily transferrable to the study of many more all-protein systems and their conformational energy landscape.
单分子Förster 共振能量转移（smFRET）是检测生物分子构象动力学的一种强大技术。虽然许多 smFRET 实验都是使用染料标记的 DNA 进行的，但这里我们描述了一种全面的方案，用于解析蛋白质系统的构象动力学 - 从质粒到数据。我们以热休克蛋白 Hsp90 为例，描述了蛋白质的生产和三倍体特异性生物偶联，使用全内反射荧光显微镜（TIRFM）进行 smFRET 测量，以及原始数据处理以揭示时间分辨的蛋白质动力学。所描述的 smFRET 方法易于转移到对许多更全蛋白质系统及其构象能量景观的研究。
