Fluorescence lifetime based distance measurement illustrates conformation changes of PYL10-CL2 upon ABA binding in solution state

Forster 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.

Chemical Synthesis of Structurally Defined Phosphorylated Ubiquitins Suggests Impaired Parkin Activation by Phosphorylated Ubiquitins with a Non-Phosphorylated Distal Unit

Mutations in genes encoding PINK1(PTEN-induced kinase 1)and Parkin(E3 ubiquitin ligase)are identified in familial Parkinson’s disease.However,it remains unclear whether the phosphorylated Ub chains activate wild-type Parkin(w-Parkin)or phosphorylated Parkin(p-Parkin),with the consequent expulsion of the damaged mitochondria.