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

Abstract

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 (K_ds) 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 K_ds by ≡50%, providing a more sensitive method. The results indicate that these bioconjugates are promising as fluorescent indicators for IP₃ quantification.