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首页> 外文期刊>Journal of Molecular Biology >Measurements of internal distance changes of the 30 S ribosome using FRET with multiple donor-acceptor pairs: Quantitative spectroscopic methods
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Measurements of internal distance changes of the 30 S ribosome using FRET with multiple donor-acceptor pairs: Quantitative spectroscopic methods

机译：使用多个供体-受体对的FRET测量30 S核糖体的内部距离变化：定量光谱法

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摘要

We present analytical and experimental procedures for determining distance changes within the 30 S subunit of the Escherichia coli ribosome using Forster resonance energy transfer (FRET). We discuss ways to contend with complexities when using FRET to measure distance changes within large multi-subunit macromolecular complexes, such as the ribosome. Complications can arise due to non-stoichiometric labeling of donor and acceptor probes, as well as environmental effects that are specific to each conjugation site. We show how to account for changes in extinction coefficients, quenching, labeling stoichiometry and other variations in the spectroscopic properties of the dye to enable more accurate calculation of distances from FRET data. We also discuss approximations that concern the orientation of the transition moments of the two dye molecules, as well as the impact of other errors in the measurement of absolute distances. Thirteen dye-pair locations with different distances using 18 independent FRET pairs conjugated to specific 30 S protein residues have been used to determine distance changes within the 30 S subunit upon association with the 50 S subunit, forming the 70 S ribosome. Here, we explain the spectroscopic methods we have used, which should be of general interest in studies that aim at obtaining quantitative distance information from FRET. (c) 2005 Elsevier Ltd. All rights reserved.
机译：我们提出了使用Forster共振能量转移（FRET）确定大肠埃希氏菌核糖体30 S亚基内距离变化的分析和实验程序。我们讨论了使用FRET测量大型多亚基大分子复合物（如核糖体）内距离变化时应对复杂性的方法。由于供体探针和受体探针的化学计量不正确，以及每个偶联位点特有的环境影响，可能会导致并发症。我们展示了如何解决消光系数的变化，猝灭，标记化学计量以及染料光谱性质的其他变化，从而能够更准确地计算FRET数据的距离。我们还将讨论有关两个染料分子跃迁矩的方向以及绝对距离测量中其他误差的影响的近似值。使用与特定的30 S蛋白残基偶联的18个独立FRET对，使用13个不同距离的染料对位置，以确定与50 S亚基缔合后形成30 S核糖体的30 S亚基内的距离变化。在这里，我们解释了我们使用的光谱方法，这些方法应该在旨在从FRET中获得定量距离信息的研究中具有普遍意义。 （c）2005 Elsevier Ltd.保留所有权利。

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