A fluorogenic red fluorescent protein heterodimer

Abstract

The expanding repertoire of genetically encoded biosensors constructed from variants of Aequorea victoria green fluorescent protein (GFP) enable the imaging of a variety of intracellular biochemical processes. To facilitate the imaging of multiple biosensors in a single cell, we undertook the development of a dimerization-dependent red fluorescent protein (ddRFP) that provides an alternative strategy for biosensor construction. An extensive process of rational engineering and directed protein evolution led to the discovery of a ddRFP with a K(d) of 33 μM and a 10-fold increase in fluorescence upon heterodimer formation. We demonstrate that the dimerization-dependent fluorescence of ddRFP can be used for detection of a protein-protein interaction in vitro, imaging of the reversible Ca²⁺-dependent association of calmodulin and M13 in live cells, and imaging of caspase-3 activity during apoptosis.

Figure 1. Overview of the Screening Procedures Used to Identify Heterodimeric and Fluorogenic RFPs

(A) Electrophoretic mobility shift screen for heterodimeric pairs of RFPs. (B) Replicaplating screen for fluorogenic and heterodimeric pairs of RFPs. (C) Tandem heterodimer proteolysis-based screen. See also Figure S1 and Table S1.

Figure 2. Characterization of Homo- and Heterodimeric Structure by Gel Electrophoresis

The contrast of each whole image has been adjusted to emphasize weak bands. (A) Validation of the SDS-PAGE electrophoretic mobility shift screen for pairs of protein variants that exhibit heterodimeric character. Proteins were loaded without boiling and were detected by imaging the red fluorescence. Lane 1, uninduced culture; lane 2, dTomato; lane 3, MBP-dTomato; lane 4, coexpressed dTomato and MBP-dTomato. Asterisk (*) indicates a species resulting from proteolysis of one dTomato-MBP linker. (B) Red fluorescence image of a gel used for PAGE analysis of four representative variants analyzed during screening (numbered 1–4). (C) Coomassie-stained gel of purified A₁ and B₁ variants. Samples were boiled in sample buffer prior to electrophoresis. The double asterisk (**) indicates the 19 kDa product of chromophore hydrolysis routinely observed for RFPs (Gross et al., 2000). The 11 kDa fragment was not observed.

Figure 3. Engineering Heterodimeric ddRFPs

(A) Position of the two symmetry-related salt bridges in dTomato. (B) Saturation-binding curves for A_0.4 with B_0.5 and A₁ with B₁. K_d values are 1.1 and 33 μM, respectively. Error bars are ± standard deviation for three independent experiments. See also Figure S2.

Figure 4. Characterization of ddRFPs

(A) Fluorescence image of E. coli expressing various proteins discussed in the text. A_0.4B_0.5 and A₁B₁ are tandem constructs. (B) Absorbance and fluorescence emission spectra for A_0.4 (dashed line; 10 μM), B_0.5 (gray line; 10 μM), and the equimolar mixture of A_0.4 and B_0.5 (black line; 10 μM each). (C) Absorbance and fluorescence spectra of A₁ (gray line; 20 μM) and tandem ddRFP-A₁B₁ (black line; 20 μM) before (solid line) and after (dashed line) treatment with trypsin. B₁ has baseline absorbance and fluorescence. See also Figure S3.

Figure 5. Fluorescence Detection of In Vitro Chemically Induced Dimerization and Imaging ddRFP-A₁B₁ in Live Cells

(A) Fluorescence of various combinations of FRB-A₁ (0.5 mM), FKBP-B₁ (0.5 μM), rapamycin, and dimethyl sulfoxide (DMSO) vehicle control. Fluorescence intensity is the mean integrated emission intensities for three independent experiments (± standard deviation) and corrected for background (i.e., assay buffer alone). (B) Imaging of Ca²⁺ dynamics with A₁-CaM and M13-B₁ in HeLa cells. Transfected HeLa cells were treated with histamine, followed by EGTA/ionomycin (abbreviated as ion) and Ca²⁺/ionomycin. (C) Imaging of caspase-3 activity with a ddRFP-A₁B₁ tandem heterodimer with a DEVD substrate in the linker region. Transfected HeLa cells were treated with TNF-α (t = 0) to initiate apoptosis, and red fluorescence was imaged as a function of time.

Figure 6. Structural Model of ddRFP-A₁B₁ Based on the AC Dimer of DsRed

The amino acid side chains for all mutations in ddRFP-A₁B₁ are shown in stick representation (Yarbrough et al., 2001). Figure was generated with PyMOL (http://www.pymol.org). See also Figure S4.