Single-Chain Lanthanide Luminescence Biosensors for Cell-Based Imaging and Screening of Protein-Protein Interactions

Abstract

Lanthanide-based, Förster resonance energy transfer (LRET) biosensors enabled sensitive, time-gated luminescence (TGL) imaging or multiwell plate analysis of protein-protein interactions (PPIs) in living cells. We prepared stable cell lines that expressed polypeptides composed of an alpha helical linker flanked by a Tb(III) complex-binding domain, GFP, and two interacting domains at each terminus. The PPIs examined included those between FKBP12 and the rapamycin-binding domain of m-Tor (FRB) and between p53 (1-92) and HDM2 (1-128). TGL microscopy revealed dramatic differences (>500%) in donor- or acceptor-denominated, Tb(III)-to-GFP LRET ratios between open (unbound) and closed (bound) states of the biosensors. We observed much larger signal changes (>2,500%) and Z'-factors of 0.5 or more when we grew cells in 96- or 384-well plates and analyzed PPI changes using a TGL plate reader. The modular design and exceptional dynamic range of lanthanide-based LRET biosensors will facilitate versatile imaging and cell-based screening of PPIs.

Keywords: Biomolecular Engineering; Molecular Interaction; Molecular Spectroscopy Techniques; Sensor.

Graphical abstract

Figure 1

Single-Chain, Tb(III)-Based LRET Biosensor Design Leverages the Narrow, Multi-line Emission Spectrum and ms-Scale Excited State Lifetime of Tb(III) Complexes to Facilitate High Signal-to-Background, Time-Gated Luminescence (TGL) Detection (A) (Top) Excitation (dotted) and emission (solid) spectra of Tb(III) (cyan) and GFP (green). Colored bars show emission band pass for detecting Tb(III) and Tb(III)-to-GFP LRET signals. (Bottom) Insertion of a microsecond-scale delay between pulsed excitation and detection enables background-free detection of Tb(III) luminescence and Tb(III)-to-GFP LRET-sensitized emission. (B) (Top) Biosensor design. An ER/K helix motif (length 10, 20, or 30 nm) separates LRET partners and affinity pairs. (Bottom) In the absence of interaction, the ER/K helix maintains affinity and detection elements far apart, ensuring low baseline LRET signal. Stochastic breaking of helix linker permits close approach and binding of affinity domains.

Figure 2

Heterodimers of Trimethoprim (TMP) and Luminescent Tb(III) Complexes Label Sensors Via Stable Binding to Escherichia Coli Dihydrofolate Reductase (eDHFR) Domains and Serve as Effective LRET Donors Compounds used in this study and the photophysical properties of their Tb(III) complexes including longest wavelength absorption maximum (λ_max), absorption coefficient at λ_max (ϵ), overall quantum yield (φ_Overall), metal-centered quantum yield (φ_Tb), lifetime (τ), and Förster distance with EGFP (R_0,EGFP) are as follows: 1. TMP-Lumi4-R₉: λ_max, 340 nm; ε, 21,000 M⁻¹cm⁻¹; φ_Overall, 0.6; φ_Tb, 0.7; τ, 2.4 ms; R_0,EGFP, 0.48 nm. 2. TMP-TTHA-cs124: λ_max, 341 nm; ϵ, 10,000 M⁻¹cm⁻¹; φ_Overall, 0.21; φ_Tb, 0.6; τ, 1.6 ms; R_0,EGFP, 0.47 nm. 3. TMP-Lumi4: photophysical properties same as for TMP-Lumi4-R₉.

Figure 3

Time-Gated Luminescence Microscopy Enables Two-Channel, Ratiometric Imaging of LRET PPI Biosensors with High Dynamic Range (A) Representative images of NIH3T3 fibroblasts stably expressing FRB-eDHFR-(ER/K)₂₀-GFP-FKBP12 approximately 20 min after stimulation with rapamycin (1 μM). Micrographs: CW GFP, steady-state GFP fluorescence (λ_ex, 480 ± 20 nm; λ_em, 535 nm ± 25 nm); TG Tb, time-gated Tb(III) luminescence (λ_ex, 365 nm, λ_em, 620 nm ± 10 nm, gate delay 10 μs); TG LRET, time-gated Tb(III)-to-GFP sensitized emission (λ_ex, 365 nm; λ_em, 520 ± 10 nm, gate delay 10 μs). Scale bars, 20 μm. TG Tb and TG LRET channel images were rendered at identical contrast. (B) Color maps of the same cells shown in (A) depict the ratio of the TG LRET image to the TG-Tb image at various time points following rapamycin stimulation. (C) Biosensor dynamic ranges increase with the length of ER/K linker due to reduction in baseline, or Off-State LRET signals. Bar graphs depict the mean, pixel-wise LRET/Tb or LRET/GFP ratios measured in regions of interest drawn within images of cells acquired before and 25 min after addition of rapamycin. Values given are averaged from 10 or more cells for each condition. Error bars, SEM.

Figure 4

TGL Analysis Robustly Detects FKBP12/FRB Interaction and Its Inhibition Following Permeabilzation of Sensor-Expressing Cells Grown in Multiwell Plates (A–D) NIH 3T3 fibroblast cells expressing FKBP/FRB sensors with varied ER/K linker lengths were seeded into 96-well (A and C) or 384-well (B and D) plates at cell densities of 40,000 or 8,000 cells/well, respectively. Following overnight incubation, cells were treated with lysis buffer containing TMP-TTHA-cs124 (25 nM). Time-gated emission signals (gate delay, 0.2 ms) at 520 nm (Tb-to-GFP LRET) and 620 nm (Tb only) were measured using a time-resolved fluorescence plate reader. (A and B) LRET/Tb ratios were substantially larger when lysis buffer contained rapamycin (1 μM). (C and D) Cells were treated with lysis buffer containing TMP-TTHA-cs124 (25 nM) and rapamycin (0.33 μM). Time-gated signals were then measured as in (A and B). Addition of ascomycin (20 μM) to lysis buffer decreased LRET/Tb emission ratios by more than 60% for all sensor linker lengths in both 96-well and 384-well plates. Bar graphs depict mean LRET/Tb ratio measured for positive controls (n = 16) and for negative controls (n = 8). Error bars, SD.

Figure 5

Large Reductions in LRET/Tb Are Observed Microscopically and in Multiwell Plates When Nutlin-3 Inhibits p53/HDM2 Interaction (A) HeLa cells stably expressing p53(1–92)-eDHFR-(ER/K)₂₀-GFP-HDM2(1–128) were imaged as in Figure 3. Representative images show diminished LRET/Tb ratio in cells that were incubated with media containing Nutlin-3 (10 μM). (B) Bar graphs depict the mean, pixel-wise LRET/Tb ratio measured in regions of interest drawn within cells. Values given are averaged from 10 or more cells for each condition. Error bars, SD. (C–E) HeLa cells expressing p53/HDM2 sensor were grown in 96-well (C and E) or 384-well (D) plates. (C and D) Time-gated measurements were obtained following overnight induction of biosensor expression with doxycycline and addition of lysis buffer containing TMP-TTHA-cs124 (2, 50 nM) and Nutlin-3 (10 μM, positive controls) or DMSO (0.25%, negative controls). (E) Cells expressing biosensor were incubated in medium containing cell-permeable Tb complex, TMP-Lumi4-R₉ (10 μM, RT, 30 min), washed 1X PBS, and incubated in PBS containing either DMSO (negative controls) or Nutlin-3 (10 μM, positive controls). Time-gated signals were then recorded. Bar graphs depict mean LRET/Tb ratio measured for positive controls (n = 16) and negative controls (n = 16 for D and E and 8 for C). Error bars, SD.