Labeling of fusion proteins with synthetic fluorophores in live cells

Abstract

A general approach for the sequential labeling of fusion proteins of O(6)-alkylguanine-DNA alkyltransferase (AGT) with different fluorophores in mammalian cells is presented. AGT fusion proteins with different localizations in the cell can be labeled specifically with different fluorophores, and the fluorescence labeling can be used for applications such as multicolor analysis of dynamic processes and fluorescence resonance energy transfer measurements. The facile access to a variety of different AGT substrates as well as the specificity of the labeling reaction should make the approach an important tool to study protein function in live cells.

Fig. 1.

General mechanism of (A) and substrates used for (B) fluorescence labeling of AGT fusion proteins. (A) Covalent labeling of AGT fusion proteins with BG derivatives. (B) Structures of BG derivatives used for fluorescence labeling of AGT fusion proteins. The maxima in the absorbance (Abs.) and fluorescence emission spectra (Em.) of the substrates after reaction with AGT are listed; the data for BGAF, BGOG, and BGSF are those of the deacetylated molecules.

Fig. 2.

Transiently transfected CHO cells expressing various AGT fusion proteins labeled with fluorescent BG derivatives. (A) AGT-NLS₃ labeled with OG using BGOG. (B) AGT-NLS₃ labeled with TMR using BGTMR. (C) AGT-NLS₃ labeled with SF using BGSF. (D) AGT-β-Gal labeled with fluorescein using BGAF. (E) AGT labeled with fluorescein. (F) EGFP. (G) AGT-CaaX labeled with TMR. (H) AGT-α-tubulin labeled with fluorescein. (I) tsVSVG-AGT labeled with fluorescein. (Scale bars, 10 μm.)

Fig. 3.

Extracellular (A–D) and FRET (E–I) applications of AGT fusion proteins. (A–D) Transiently transfected HEK293 cells expressing AGT-NK₁. (A) AGT-NK₁ labeled with fluorescein. (B) AGT-NK₁ labeled with Cy3 using BGCy3. (C) Binding of SP-rho (50 nM) to the same cell as in A. (D) Time-resolved binding of SP-rho (50 nM) to an HEK293 cell displaying AGT-NK₁ and its replacement through the addition of unlabeled ligand (2.5 μM) at the indicated time (black arrow). (E–I) Transiently transfected CHO cells expressing AGT-EGFP-NLS₃ labeled with SF for FRET detection. (E) Overlay of transmission and fluorescence channel for EGFP before labeling with SF (excitation 488 nm/emission 505–530 nm). (F) Fluorescence channel for SF before labeling with SF (excitation 488 nm/emission > 650 nm). (G) Fluorescence channel for EGFP 60 min after labeling with SF (excitation 488 nm/emission 505–530 nm). (H) Overlay of transmission and fluorescence channel for SF after labeling with SF (excitation 488 nm/emission > 650 nm). (I) Relative fluorescence intensities (RFI) of the EGFP channel before and after labeling with SF. Columns E and G correspond to the fluorescence intensities (RFI) of the EGFP channel of the cell shown in E and G. Columns J and K correspond to the fluorescence intensities (RFI) of the EGFP channel of a cell before and after incubation with SF but previously incubated with BG (20 μM). (Scale bars 10 μm.)

Fig. 4.

Multicolor analysis of tsVSVG-AGT and labeling of AGT-CaaX in cells possessing endogenous AGT. (A–C) Sequential labeling of tsVSVG-AGT: Labeling with fluorescein at permissive temperature (34°C) and with SF at nonpermissive temperature (40°C). (A) Overlay of transmission and fluorescence micrographs. (B) Fluorescence channel for fluorescein-labeled tsVSVG-AGT (excitation 488 nm/emission 505–530 nm). (C) Fluorescence channel for SF-labeled tsVSVG-AGT (excitation 543 nm/emission > 650 nm). (D and E) Labeling of AGT-CaaX in HEK293 cells possessing endogenous AGT. (D) Fluorescence channel after labeling with fluorescein (excitation 488 nm/emission 505–530 nm). (E) Transmission channel of same cells as in G. (Scale bar, 10 μm.)