Long-distance tmFRET using bipyridyl- and phenanthroline-based ligands.
Long-distance tmFRET using bipyridyl- and phenanthroline-based ligands.
复制标题
使用联吡啶和菲咯啉配体进行长距离 tmFRET。
DOI：
10.1101/2023.10.09.561591
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发表时间：
2024
期刊：
影响因子：
--
通讯作者：
Zagotta,WilliamN
中科院分区：
文献类型：
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作者：
Gordon,SharonaE;Evans,EricGB;Otto,ShaunaC;Tessmer,MaxxH;Shaffer,KyleD;Gordon,MosheT;Petersson,EJames;Stoll,Stefan;Zagotta,WilliamN
在过去的十年里，在确定蛋白质结构方面取得了巨大的进展，人们重新认识到结构必须与动力学和能量学相结合才能理解功能。荧光光谱学，特别是Förster共振能量转移（FRET），由于其在生理温度下的应用和在ångström尺度上测量动力学的能力，为动力学和能量学提供了一个很好的窗口。我们最近发展了过渡金属FRET （tmFRET）来研究麦芽糖结合蛋白的变构调节，并报道了麦芽糖依赖距离变化的测量，准确度为~ 1.5 Å。当与非规范氨基酸作为供体配对时，我们之前的tmFRET受体在10到20 Å的工作距离内是有用的。在这里，我们使用半胱氨酸反应性联吡啶和邻菲罗啉化合物作为Fe2+和Ru2+的螯合剂来生产新的tmFRET受体，将工作距离扩大到50 Å，同时保留了我们解决即使是小的麦芽糖依赖性距离变化的能力。我们将测量到的FRET效率与基于供体和受体旋转集合模型的预测进行了比较，以证明使用我们的新探针对tmFRET的稳态测量具有前所未有的测量生理条件下构象重排的能力。
With the great progress on determining protein structures over the last decade comes a renewed appreciation that structures must be combined with dynamics and energetics to understand function. Fluorescence spectroscopy, specifically Förster resonance energy transfer (FRET), provides a great window into dynamics and energetics due to its application at physiological temperatures and ability to measure dynamics on the ångström scale. We have recently advanced transition metal FRET (tmFRET) to study allosteric regulation of maltose binding protein and have reported measurements of maltose-dependent distance changes with an accuracy of ∼1.5 Å. When paired with the noncanonical amino acid Acd as a donor, our previous tmFRET acceptors were useful over a working distance of 10 to 20 Å. Here, we use cysteine-reactive bipyridyl and phenanthroline compounds as chelators for Fe2+and Ru2+to produce novel tmFRET acceptors to expand the working distance to as long as 50 Å, while preserving our ability to resolve even small maltose-dependent changes in distance. We compare our measured FRET efficiencies to predictions based on models using rotameric ensembles of the donors and acceptors to demonstrate that steady-state measurements of tmFRET with our new probes have unprecedented ability to measure conformational rearrangements under physiological conditions.