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.
Project description:Protein-protein interactions (PPIs) are central to biological processes and represent an important class of therapeutic targets. Here we show that the interaction between FK506-binding protein 12 fused to green fluorescent protein (GFP-FKBP) and the rapamycin-binding domain of mTor fused to Escherichia coli dihydrofolate reductase (FRB-eDHFR) can be sensitively detected (signal-to-background ratio (S/B)>100) and accurately quantified within an impure cell lysate matrix using a luminescence resonance energy transfer (LRET) assay. Ascomycin-mediated inhibition of GFP-FKBP-rapamycin-FRB-eDHFR complex formation was also detected at high S/B ratio (>80) and Z'-factor (0.89). The method leverages the selective, stable binding of trimethoprim (TMP)-terbium complex conjugates to eDHFR, and time-resolved, background-free detection of the long-lifetime (?ms) terbium-to-GFP LRET signal that indicates target binding. TMP-eDHFR labeling can be adapted to develop high-throughput screening assays and complementary, quantitative counter-screens for a wide variety of PPI targets with a broad range of affinities that may not be amenable to purification.
Project description:When suitably labeled bulk tRNAs are transfected into cells they give rise to FRET (fluorescence resonance energy transfer) signals via binding to ribosomes that provide a measure of total protein synthesis. Application of this approach to monitoring rates of specific protein synthesis requires achieving a very high signal-to-noise ratio. Such high ratios may be attainable using LRET (luminescence resonance energy transfer) in place of FRET. Lanthanide complexes containing an antenna chromophore are excellent LRET donors. Here we describe the synthesis of a Phe-tRNA(Phe) labeled with a Tb(3+) complex, denoted Tb(3+)-Phe-tRNA(Phe) that, notwithstanding the bulkiness of the Tb(3+) complex, is active in protein synthesis.
Project description:Luminescence resonance energy transfer (LRET) offers many advantages for accurate measurements of distances between specific sites in living cells, but progress in developing a methodology for implementing this technique has been limited. We report here the design, expression, and characterization of a test protein for development of a LRET methodology. The protein, which we call DAL, contains the following domains (from the N-terminus): Escherichia coli dihydrofolate reductase (DHFR), the third and fourth ankyrin repeats of p16(INK4a), a lanthanide-binding tag (LBT), and a hexahistidine tag. LBT binds Tb(3+) with a submicromolar dissociation constant. LRET was measured from the Tb(3+) site on LBT to transition metals bound to the hexa-His tag and to fluorescein methotrexate bound to DHFR. The measured distances were consistent with a molecular model constructed from the known crystal structures of the constituent domains of DAL. The results indicate that the two C-terminal ankyrin domains of p16(INK4a) are stably folded when combined with other protein domains. We found that Tb(3+) binds to DAL in the cytoplasm of live E. coli cells, and thus, DAL is useful as an indicator for studies of metal transport. We also used DAL to measure LRET from Tb(3+) to Cu(2+) in the cytoplasm of live E. coli cells. The rates of Tb(3+) and Cu(2+) transport were not affected by a proton uncoupler or an ATP synthase inhibitor. Reversal of the membrane potential had a small inhibitory effect, and removal of lipopolysaccharide had a small accelerating effect on transport. Changing the external pH from 7 to 5 strongly inhibited the Tb(3+) signal, suggesting that the Tb(3+)-LBT interaction is useful as a cytoplasmic pH indicator in the range of approximately pH 5-6.
Project description:Integral membrane proteins play essential roles in all living systems; however, major technical hurdles challenge analyses of this class of proteins. Biophysical approaches that provide structural information to complement and leverage experimentally determined and computationally predicted structures are urgently needed. Herein we present the application of luminescence resonance energy transfer (LRET) for investigating the interactions of the polytopic membrane-bound oligosaccharyl transferases (OTases) with partner substrates. Monomeric OTases, such as the PglBs from Campylobacter jejuni and Campylobacter lari, catalyze transfer of glycans from membrane-associated undecaprenol diphosphate-linked substrates to proteins in the bacterial periplasm. LRET-based distance measurements are enabled by the inclusion of an encoded N-terminal lanthanide-binding tag (LBT), and LRET between the luminescent (LBT)-Tb(3+) donor complex and fluorescently labeled peptide and glycan substrates provides discrete distance measurements across the span of the membrane. LRET-based measurements of detergent-solubilized PglB from C. lari allowed direct comparison with the distances based on the previously reported the C. lari PglB crystal structure, thereby validating the approach in a defined system. Distance measurements between peptide and glycan substrates and the C. jejuni PglB offer new experimental information on substrate binding to the related, but structurally uncharacterized, eukaryotic OTase.
Project description:Probes and biosensors that incorporate luminescent Tb(III) or Eu(III) complexes are promising for cellular imaging because time-gated microscopes can detect their long-lifetime (approximately milliseconds) emission without interference from short-lifetime (approximately nanoseconds) fluorescence background. Moreover, the discrete, narrow emission bands of Tb(III) complexes make them uniquely suited for multiplexed imaging applications because they can serve as Förster resonance energy transfer (FRET) donors to two or more differently colored acceptors. However, lanthanide complexes have low photon emission rates that can limit the image signal/noise ratio, which has a square-root dependence on photon counts. This work describes the performance of a wide-field, time-gated microscope with respect to its ability to image Tb(III) luminescence and Tb(III)-mediated FRET in cultured mammalian cells. The system employed a UV-emitting LED for low-power, pulsed excitation and an intensified CCD camera for gated detection. Exposure times of ?1 s were needed to collect 5-25 photons per pixel from cells that contained micromolar concentrations of a Tb(III) complex. The observed photon counts matched those predicted by a theoretical model that incorporated the photophysical properties of the Tb(III) probe and the instrument's light-collection characteristics. Despite low photon counts, images of Tb(III)/green fluorescent protein FRET with a signal/noise ratio ? 7 were acquired, and a 90% change in the ratiometric FRET signal was measured. This study shows that the sensitivity and precision of lanthanide-based cellular microscopy can approach that of conventional FRET microscopy with fluorescent proteins. The results should encourage further development of lanthanide biosensors that can measure analyte concentration, enzyme activation, and protein-protein interactions in live cells.
Project description:A quinoline sensitizer-centered lanthanide chelate system of novel design for TR-LRET was prepared; it exhibited high labelling efficiency with a his-tagged protein (ERalpha-LBD) on the Ni-NTA beads, using a mixed metal chelate protocol, and it functioned well in TR-LRET protein-protein interaction assays.
Project description:Voltage-gated sodium channels (Navs) play crucial roles in excitable cells. Although vertebrate Nav function has been extensively studied, the detailed structural basis for voltage-dependent gating mechanisms remain obscure. We have assessed the structural changes of the Nav voltage sensor domain using lanthanide-based resonance energy transfer (LRET) between the rat skeletal muscle voltage-gated sodium channel (Nav1.4) and fluorescently labeled Nav1.4-targeting toxins. We generated donor constructs with genetically encoded lanthanide-binding tags (LBTs) inserted at the extracellular end of the S4 segment of each domain (with a single LBT per construct). Three different Bodipy-labeled, Nav1.4-targeting toxins were synthesized as acceptors: ?-scorpion toxin (Ts1)-Bodipy, KIIIA-Bodipy, and GIIIA-Bodipy analogs. Functional Nav-LBT channels expressed in Xenopus oocytes were voltage-clamped, and distinct LRET signals were obtained in the resting and slow inactivated states. Intramolecular distances computed from the LRET signals define a geometrical map of Nav1.4 with the bound toxins, and reveal voltage-dependent structural changes related to channel gating.
Project description:Large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels are involved in a large variety of physiological processes. Regulatory β-subunits are one of the mechanisms responsible for creating BK channel diversity fundamental to the adequate function of many tissues. However, little is known about the structure of its voltage sensor domain. Here, we present the external architectural details of BK channels using lanthanide-based resonance energy transfer (LRET). We used a genetically encoded lanthanide-binding tag (LBT) to bind terbium as a LRET donor and a fluorophore-labeled iberiotoxin as the LRET acceptor for measurements of distances within the BK channel structure in a living cell. By introducing LBTs in the extracellular region of the α- or β1-subunit, we determined (i) a basic extracellular map of the BK channel, (ii) β1-subunit-induced rearrangements of the voltage sensor in α-subunits, and (iii) the relative position of the β1-subunit within the α/β1-subunit complex.
Project description:Complex patterns of protein-protein interactions (PPInts) are involved in almost all cellular processes. This has stimulated the development of a wide range of methods to characterize PPInts in detail. Methods with fluorescence resonance energy transfer can be technically challenging and suffer from several limitations, which could be overcome by switching to luminescence resonance energy transfer (LRET) with lanthanide ions such as Tb3+. With LRET, energy transfer between PPInt partners works over a larger distance and with less topological constraints; moreover, the long-lived luminescence of lanthanides allows one to bypass the short-lived background fluorescence. We have developed a novel LRET method to investigate PPInts between partners expressed as fusion proteins with genetically encoded donor and acceptor moieties. Upon UV excitation of a tryptophan within a lanthanide binding peptide, the Tb3+ luminescence is harnessed to excite either a green or a red fluorescent protein. We demonstrate the usefulness of the LRET assay by applying it to analyze the interactions of the molecular chaperones HSP70 and HSP90 with their common co-chaperone HOP/Sti1. We recapitulate the previously described interaction specificities between the HSP70/HSP90 C-termini and tetratricopeptide repeat domains of HOP/Sti1 and demonstrate the impact of single point mutants on domain-domain interactions.
Project description:Fluorescence lifetime measurements can provide quantitative readouts of local fluorophore environment and can be applied to biomolecular interactions via Förster resonant energy transfer (FRET). Fluorescence lifetime imaging (FLIM) can therefore provide a high content analysis (HCA) modality to map protein-protein interactions (PPIs) with applications in drug discovery, systems biology and basic research. We present here an automated multiwell plate reader able to perform rapid unsupervised optically sectioned FLIM of fixed and live biological samples and illustrate its potential to assay PPIs through application to Gag protein aggregation during the HIV life cycle. We demonstrate both hetero-FRET and homo-FRET readouts of protein aggregation and report the first quantitative evaluation of a FLIM HCA assay by generating dose response curves through addition of an inhibitor of Gag myristoylation. Z' factors exceeding 0.6 are realised for this FLIM FRET assay.