开发多通道 smFRET 方法以剖析核糖体机制

Ran Lin,  and , Yuhong Wang*, 
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引用次数: 0

摘要

核糖体是一种直径约为 20 纳米的 2.6 兆道尔顿生物大分子,它协调众多配体、因子和调节器,以高保真和高速度翻译蛋白质。要了解它的复杂功能,就必须进行多视角观测。我们开发了一种双-FRET 单分子佛尔斯特共振能量转移方法(dual-smFRET),可在 10 秒的时间窗口内同时观察和关联同一复合体中 tRNA 的动态和伸长因子 G(EF-G)的构象。通过同步激光快门和电动滤波器组,可在连续 5 秒的时间间隔内捕获两个 FRET 信号,时间间隔为 50-100 毫秒。我们观察到单标记、双标记和四标记核糖体复合物发出不同的荧光。通过全面的光谱分析和校正,我们区分并关联了核糖体两个部分的构象变化,为研究核糖体在易位过程中的协调和时间提供了更多的视角。我们的装置具有多功能性,可容纳多达六对 FRET,这为研究大型生物分子和各种生物系统提供了更广泛的应用前景。
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Developing Multichannel smFRET Approach to Dissecting Ribosomal Mechanisms

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.

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来源期刊
Chemical & Biomedical Imaging
Chemical & Biomedical Imaging 化学与生物成像-
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期刊介绍: Chemical & Biomedical Imaging is a peer-reviewed open access journal devoted to the publication of cutting-edge research papers on all aspects of chemical and biomedical imaging. This interdisciplinary field sits at the intersection of chemistry physics biology materials engineering and medicine. The journal aims to bring together researchers from across these disciplines to address cutting-edge challenges of fundamental research and applications.Topics of particular interest include but are not limited to:Imaging of processes and reactionsImaging of nanoscale microscale and mesoscale materialsImaging of biological interactions and interfacesSingle-molecule and cellular imagingWhole-organ and whole-body imagingMolecular imaging probes and contrast agentsBioluminescence chemiluminescence and electrochemiluminescence imagingNanophotonics and imagingChemical tools for new imaging modalitiesChemical and imaging techniques in diagnosis and therapyImaging-guided drug deliveryAI and machine learning assisted imaging
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