High-contrast and real-time visualization of membrane proteins in live cells with malachite green-based fluorogenic probes

Imaging dynamics of membrane proteins of live cells in a wash-free and real-time manner has been a challenging task. Herein, we report unprecedented applications of malachite green (MG), an organic dye widely used in pigment industry, as a switchable fluorophore to monitor membrane enzymes or noncatalytic proteins in live cells. Conformationally flexible MG is non-fluorescent in aqueous solution, yet covalent binding with endogenous proteins of cells significantly enhances its fluorescence at 670 nm by restricting flexibility of dye. Integrating a phosphate-caged quinone methide precursor with MG yielded a covalent labeling fluorogenic probe, allowing real-time imaging of membrane alkaline phosphatase (ALP, a model catalytic protein) activity in live cells with over 100-fold enhancement of fluorescence intensity. Moreover, MG is also applicable to image non-catalytic protein by conjugation with protein-specific ligand. A fluorogenic probe consisted of c-RGDfK peptide and MG proved to be compatible with wash-free and real-time visualization of non-catalytic integrin alpha(v)beta(3) in live cells with high contrast. (C) 2021 Published by Elsevier B.V. on behalf of Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.

Automated summary

This study presents the unprecedented applications of malachite green as a switchable fluorophore for real-time imaging of membrane enzymes and noncatalytic proteins in live cells. The fluorescence intensity of malachite green can be significantly enhanced by covalent binding with endogenous proteins, enabling the monitoring of enzyme activities and protein visualization. The findings highlight the potential of malachite green as a versatile tool for studying membrane protein dynamics.