本文主要研究内容
作者（2019）在《Fluorescence lifetime based distance measurement illustrates conformation changes of PYL10-CL2 upon ABA binding in solution state》一文中研究指出：F?rster 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.
Abstract
F?rster 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.
论文参考文献
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：Fluorescence lifetime based distance measurement illustrates conformation changes of PYL10-CL2 upon ABA binding in solution state论文
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