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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Short-distance probes for protein backbone structure based on energy transfer between bimane and transition metal ions
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Short-distance probes for protein backbone structure based on energy transfer between bimane and transition metal ions

机译：基于Bimane和过渡金属离子之间的能量转移的蛋白质骨架结构的短距离探针

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摘要

The structure and dynamics of proteins underlies the workings of virtually every biological process. Existing biophysical methods are inadequate to measure protein structure at atomic resolution, on a rapid time scale, with limited amounts of protein, and in the context of a cell or membrane. FRET can measure distances between two probes, but depends on the orientation of the probes and typically works only over long distances comparable with the size of many proteins. Also, common probes used for FRET can be large and have long, flexible attachment linkers that position dyes far from the protein backbone. Here, we improve and extend a fluorescence method called transition metal ion FRET that uses energy transfer to transition metal ions as a reporter of short-range distances in proteins with little orientation dependence. This method uses a very small cysteine-reactive dye monobromobimane, with virtually no linker, and various transition metal ions bound close to the peptide backbone as the acceptor. We show that, unlike larger f luorophores and longer linkers, this donor-acceptor pair accurately reports short-range distances and changes in backbone distances. We further extend the method by using cysteine-reactive metal chelators, which allow the technique to be used in protein regions of unknown secondary structure or when native metal ion binding sites are present. This improved method overcomes several of the key limitations of classical FRET for intramolecular distance measurements.
机译：蛋白质的结构和动力学是几乎每个生物过程工作的基础。现有的生物物理方法不足以在有限的蛋白质量下以及在细胞或膜的情况下以快速的时间尺度以原子分辨率测量蛋白质结构。 FRET可以测量两个探针之间的距离，但取决于探针的方向，通常只能在与许多蛋白质大小相当的长距离上工作。同样，用于FRET的普通探针可能很大，并且具有长而灵活的连接接头，从而使染料远离蛋白质骨架。在这里，我们改进和扩展了一种称为过渡金属离子FRET的荧光方法，该方法利用能量转移将过渡金属离子作为蛋白质中短程距离的报告分子，几乎没有方向依赖性。该方法使用非常小的半胱氨酸反应性染料单溴二烯，几乎没有接头，结合肽主链附近的各种过渡金属离子作为受体。我们表明，与较大的荧光团和较长的接头不同，该供体-受体对准确报告了短距离距离和主链距离的变化。我们通过使用半胱氨酸反应性金属螯合剂进一步扩展了该方法，这使该技术可用于未知二级结构的蛋白质区域或存在天然金属离子结合位点的蛋白质区域。这种改进的方法克服了传统FRET在分子内距离测量中的几个关键限制。

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