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首页> 外文期刊>Biochemistry >OS-FRET: A New One-Sample Method for Improved FRET Measurements
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OS-FRET: A New One-Sample Method for Improved FRET Measurements

机译：OS-FRET：一种新的单样本方法，用于改善FRET测量

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Fluorescence resonance energy transfer (FRET) is a powerful tool for studying macromolecularnassemblies in vitro under near-physiological conditions. Here we present a new type of one-sample FRETn(OS-FRET) method employing a novel, nonfluorescent methanethiosulfonate-linked acceptor that can benreversibly coupled to a target sulfhydryl residue via a disulfide bond. After the quenched donor emission isnquantitated, the acceptor is removed by reduction, allowing measurement of unquenched donor emission innthe same sample. Previous one-sample methods provide distinct advantages in specific FRET applications.nThe new OS-FRET method is a generalizable spectrochemical approach that can be applied to macro-nmolecular systems lacking essential disulfide bonds and eliminates the potential systematic errors of somenearlier one-samplemethods. In addition, OS-FRET enables quantitative FRETmeasurements in virtually anynfluorescence spectrometer or detection device. Compared to conventional multisample FRET methods, OS-nFRET conserves sample, increases the precision of data, and shortens the time permeasurement. The utility ofnthe method is illustrated by its application to a protein complex of known structure formed by CheWand thenP4-P5 fragment of CheA, both from Thermotoga maritima. The findings confirm the practicality andnadvantages ofOS-FRET.Anticipated applications ofOS-FRET include analysis ofmacromolecular structure,nbinding and conformational dynamics, and high-throughput screening for interactions and inhibitors.
机译：荧光共振能量转移（FRET）是研究近乎生理条件下的体外大分子组装的有力工具。在这里，我们介绍一种新型的单样本FRETn（OS-FRET）方法，该方法采用了一种新型的，非荧光的甲硫代磺酸盐连接的受体，该受体可以通过二硫键不可逆地偶联至目标巯基残基。量化淬灭的供体发射后，通过还原去除受体，从而可以测量同一样品中未淬灭的供体发射。以前的一样品方法在特定的FRET应用中具有明显的优势.n新的OS-FRET方法是一种可推广使用的光谱化学方法，可用于缺乏必要的二硫键的大分子系统，并消除了较早的一样品方法的潜在系统误差。此外，OS-FRET几乎可以在任何荧光光谱仪或检测设备中进行定量FRET测量。与传统的多样本FRET方法相比，OS-nFRET可节省样本，提高数据精度并缩短每次测量的时间。该方法的实用性通过将其应用于已知结构的蛋白质复合物来说明，所述蛋白质复合物由CheW和随后来自CheritA的P4-P5片段形成，所述片段均来自Maritoma。这些发现证实了OS-FRET的实用性和优越性。OS-FRET的预期应用包括大分子结构分析，结合和构象动力学以及高通量筛选相互作用和抑制剂。

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