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 non-catalytic 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 α_vβ₃ in live cells with high contrast.