Fluorescence lifetime based distance measurement illustrates conformation changes of PYL10-CL2 upon ABA binding in solution state
Fluorescence lifetime based distance measurement illustrates conformation changes of PYL10-CL2 upon ABA binding in solution state
复制标题
基于荧光寿命的距离测量说明了溶液状态下 ABA 结合后 PYL10-CL2 的构象变化
DOI：
10.1016/j.cclet.2019.01.020
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发表时间：
2019-05-01
影响因子：
9.1
通讯作者：
Tian，Changlin
中科院分区：
文献类型：
--
作者：
Zhou，Peng;Lv，Pei;Tian，Changlin
Förster共振能量转移（FRET）是一种广泛使用的距离测量方法来说明蛋白质构象动力学。FRET方法依赖于供体和受体之间的距离，以及标记效率，荧光团的大小和性质。在这里，我们标记了一对小的荧光团，并通过荧光寿命分析计算能量转移效率，这可以提供比强度衰减更可靠的距离测量。供体荧光团，7-羟基香豆素-4-基-乙基甘氨酸（HC），被遗传纳入PYL 10的特定位点，获得完整的标记效率。受体荧光团Alexa 488通过二硫键标记，其标记效率通过吸收峰和寿命群体来估计。荧光寿命和各向异性分析表明，ABA诱导的局部构象变化和动力学的几个HC掺入位点的PYL 10。基于寿命的FRET距离测量说明了PYL 10的构象变化与阿坝的应用或不应用，这与先前报道的晶体结构一致。
Förster resonance energy transfer (FRET) is a widely used distance measurement method to illustrate protein conformational dynamics. The FRET method relies on the distance between donor and acceptor, as well as the labelling efficiency, the size and the properties of the fluorophores. Here, we labelled a pair of small fluorophores and calculated the energy transferred efficiency through fluorescence lifetime analysis, which can provide more reliable distance measurement than intensity attenuation. The donor fluorophore, 7-hydroxycoumarin-4-yl-ethylglycine (HC), was genetically incorporated into specific sites of PYL10, obtaining complete labelling efficiency. The acceptor fluorophore, Alexa488, was labelled through the disulfide bond, whose labelling efficiency was estimated through both absorption peaks and lifetime populations. Fluorescence lifetime and anisotropy analysis showed ABA-induced local conformation changes and dynamics of several HC incorporation sites of PYL10. The lifetime-based FRET distance measurement illustrated the conformation changes of PYL10 with or without ABA application, which is consistent with the previously reported crystal structures.