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

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.

Keywords: hCRBPII; imaging; merocyanine; protein engineering; red fluorescence.

Figure 1.

Merocyanine aldehyde 1 binds to an active site Lys residue to generate a red-shifted cyanine dye (POI = protein of interest).

Figure 2.

Live-cell imaging of HeLa cells expressing EGFP-hCRBPII_tetra (N-terminus attachment of hCRBPII) after incubation with 0.25 μM MCRA for 1 h at 37 °C followed by additional incubation in MCRA-free media for 0 min, 2 h, 4 h and 8 h. Live-cell imaging in HeLa cells expressing EGFP-hCRBPII_tetra^Q108L (N-terminus attachment of hCRBPII) serves as a control experiment without the reactive lysine. All samples were washed with PBS buffer twice before imaging. All confocal images are pseudocolored. All images were collected with a 40x oil immersed objective. MCRA/hCRBPII images in red were recorded using 594 nm laser and longpass (LP) filter 615 nm; all images in green were recorded with 488 nm laser and bandpass (BP) filter 505–530 nm. Scale bars are 20 μm.

Figure 3.

Live-cell imaging of the COS-7, U2OS and HeLa cells expressing hCRBPII_nona in the cytosol (EGFP-hCRBPII_nona, N-terminus attachment of hCRBPII), labelled with 0.25 μM MCRA for 30 seconds. HeLa cells expressing EGFP-hCRBPII_nona-3NLS and hCRBPII_nona-NES were also labelled with 0.25 μM MCRA for 30 seconds (NLS and NES attached to C-terminus of hCRBPII). All samples were washed with PBS buffer twice and immediately imaged. All confocal images are pseudocolored. All images were collected with a 40x oil immersed objective. MCRA/hCRBPII images in red were recorded using 594 nm laser and LP filter 615 nm; all images in green were recorded with 488 nm laser and BP 505–530 nm. Scale bars are 20 μm.

Figure 4.

Imaging of yeast cells expressing Fex1p fusion proteins; a. N-EGFP-Fex1-C; b. N-Fex1-EGFP-C; and c. N-Fex1-hCRBPII_nona-C. Cells expressing N-Fex1-hCRBPII_nona-C were incubated with 0.5 μM MCRA for 10 seconds, washed with water five times and then imaged. All confocal images are pseudocolored. All images were collected with a widefield microscope under white light. Scale bars are 10 μm.