Conformational changes of PYL10-Cl2 in solution with ABA

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.