Detection of vimentin serine phosphorylation by multicolor Quenchbodies

Protein phosphorylation is a key event in intracellular signal transduction, and fluorescent biosensor for the specific phosphorylation event in a target protein is considered highly useful as a tool of cellular biology and drug screening. Vimentin, the most abundant intermediate filament protein, is phosphorylated at its specific serine (Ser) residues in a cell cycle dependent manner. Its structural and functional characteristics are modified by the phosphorylation, which affects biological properties of the cell. Here we present the detection of the vimentin Ser71 phosphorylation (PS71) and the vimentin Ser82 phosphorylation (PS82) using a novel fluorescent biosensor Quenchbody, which works on the principle of antigen-dependent removal of a quenching effect by intrinsic tryptophan residues on a carboxytetramethylrhodamine (TAMRA) dye incorporated at the N-terminal region of single chain antibody variable region. First, we found that rhodamine 6G (R6G)-labeled Quenchbody shows superior response than TAMRA-labeled one. Next, we made several Quenchbodies to detect PS71 and PS82. After optimization of reaction conditions, the fluorescence intensity of V(H)-V(L) type PS71 Quenchbody labeled with R6G at two positions was increased to 4.0-fold in an antigen dependent manner. Furthermore, the fluorescence intensity of doubly R6G-labeled V(L)-V(H) type PS82 Quenchbody was increased to 6.7-fold immediately after adding antigen peptide, also suggesting deeper quenching due to H-dimer formation between the dyes. Due to its simplicity, the Quenchbody-based phosphorylation biosensors will be widely applicable to in vitro diagnostics, drug screening and imaging in a rapid, simple and high-sensitive manner.