Fluorescent indicators for inositol 1,4,5-trisphosphate based on bioconjugates of pleckstrin homology domain and fluorescent dyes

Bioconjugates of fluorescent dyes and the recombinant pleckstrin homology (PH) domain of phospholipase Cδ1 were produced with the aim of developing a method to quantify inositol 1,4,5-trisphosphate (IP3) in biological samples. We replaced Cys-96 of the PH domain with Ser while retaining Cys-48 to which thiol-reactive fluorescent dyes can be coupled specifically. Acrylodan- and Dapoxyl-labelled C96S PH domain mutants exhibited fluorescence upon UV illumination with an emission peak at wavelengths of 505 and 514 nm, respectively. IP3 induced decreases in the fluorescence intensity with a red shift in the emission spectra. The dissociation constants (Kds) of the acrylodan- and Dapoxyl-labelled PH domains for IP3 were 659 and 586 nM, respectively. An additional mutation (C96S/V58K) in the PH domain decreased the Kds by ≡50%, providing a more sensitive method. The results indicate that these bioconjugates are promising as fluorescent indicators for IP3 quantification.