Lin Ran, Wang Yuhong
Department of Biology and Biochemistry, University of Houston, Houston, Texas 77204, United States.
Chem Biomed Imaging. 2024 Mar 21;2(7):501-509. doi: 10.1021/cbmi.4c00010. eCollection 2024 Jul 22.
The ribosome, a 2.6 megadalton biomolecule measuring approximately 20 nm in diameter, coordinates numerous ligands, factors, and regulators to translate proteins with high fidelity and speed. Understanding its complex functions necessitates multiperspective observations. We developed a dual-FRET single-molecule Förste Resonance Energy Transfer method (dual-smFRET), allowing simultaneous observation and correlation of tRNA dynamics and Elongation Factor G (EF-G) conformations in the same complex, in a 10 s time window. By synchronizing laser shutters and motorized filter sets, two FRET signals are captured in consecutive 5 s intervals with a time gap of 50-100 ms. We observed distinct fluorescent emissions from single-, double-, and quadruple-labeled ribosome complexes. Through comprehensive spectrum analysis and correction, we distinguish and correlate conformational changes in two parts of the ribosome, offering additional perspectives on its coordination and timing during translocation. Our setup's versatility, accommodating up to six FRET pairs, suggests broader applications in studying large biomolecules and various biological systems.
核糖体是一种直径约20纳米、分子量为2.6兆道尔顿的生物分子，它协调众多配体、因子和调节因子，以高保真度和速度翻译蛋白质。要理解其复杂功能需要多视角观察。我们开发了一种双荧光共振能量转移单分子Förster共振能量转移方法（双单分子荧光共振能量转移法），能够在10秒的时间窗口内，对同一复合物中的tRNA动态和延伸因子G（EF-G）构象进行同步观察和关联。通过同步激光快门和电动滤光片组，以50 - 100毫秒的时间间隔，在连续的5秒间隔内捕获两个荧光共振能量转移信号。我们观察到单标记、双标记和四标记核糖体复合物发出的不同荧光。通过全面的光谱分析和校正，我们区分并关联了核糖体两部分的构象变化，为其在转位过程中的协调和时间安排提供了更多视角。我们的装置具有通用性，可容纳多达六对荧光共振能量转移对，这表明其在研究大型生物分子和各种生物系统方面有更广泛的应用。
