Engineering of a Red Fluorogenic Protein/Merocyanine Complex for Live-Cell Imaging

Graphical Abstract

Seeing red rapidly: A small cytosolic protein, human cellular retinol binding protein II, has been engineered to bind merocyanine with high binding affinity and with rapid kinetics, resulting in a red-fluorescent protein complex. Its use as a protein tag in live cell imaging is demonstrated.

Abstract

A reengineered human cellular retinol binding protein II (hCRBPII), a 15-kDa protein belonging to the intracellular lipid binding protein (iLBP) family, generates a highly fluorescent red pigment through the covalent linkage of a merocyanine aldehyde to an active site lysine residue. The complex exhibits “turn-on” fluorescence, due to a weakly fluorescent aldehyde that “lights up” with subsequent formation of a strongly fluorescent merocyanine dye within the binding pocket of the protein. Cellular penetration of merocyanine is rapid, and fluorophore maturation is nearly instantaneous. The hCRBPII/merocyanine complex displays high quantum yield, low cytotoxicity, specificity in labeling organelles, and compatibility in both cancer cell lines and yeast cells. The hCRBPII/merocyanine tag is brighter than most common red fluorescent proteins.