Antibody engineering-driven controllable chemiluminescence resonance energy transfer for immunoassay with tunable dynamic range

G protein–coupled receptors (GPCRs) are prominent targets to new therapeutics for a range of diseases. Comprehensive assessments of their cellular interactions with bioactive compounds, particularly in a kinetic format, are imperative to the development of drugs with improved efficacy. Hence, we developed complementary cellular assays that enable equilibrium and real-time analyses of GPCR ligand engagement and consequent activation, measured as receptor internalization. These assays utilize GPCRs genetically fused to an N-terminal HiBiT peptide (1.3 kDa), which produces bright luminescence upon high-affinity complementation with LgBiT, an 18-kDa subunit derived from NanoLuc. The cell impermeability of LgBiT limits signal detection to the cell surface and enables measurements of ligand-induced internalization through changes in cell-surface receptor density. In addition, bioluminescent resonance energy transfer is used to quantify dynamic interactions between ligands and their cognate HiBiT-tagged GPCRs through competitive binding with fluorescent tracers. The sensitivity and dynamic range of these assays benefit from the specificity of bioluminescent resonance energy transfer and the high signal intensity of HiBiT/LgBiT without background luminescence from receptors present in intracellular compartments. These features allow analyses of challenging interactions having low selectivity or affinity and enable studies using endogenously tagged receptors. Using the β-adrenergic receptor family as a model, we demonstrate the versatility of these assays by utilizing the same HiBiT construct in analyses of multiple aspects of GPCR pharmacology. We anticipate that this combination of target engagement and proximal functional readout will prove useful to the study of other GPCR families and the development of new therapeutics.

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Design of fluorescence resonance energy transfer (FRET)-based cGMP indicators: a systematic approach

Biochemical Journal ◽

10.1042/bj20070348 ◽

2007 ◽

Vol 407 (1) ◽

pp. 69-77 ◽

Cited By ~ 95

Author(s):

Michael Russwurm ◽

Florian Mullershausen ◽

Andreas Friebe ◽

Ronald Jäger ◽

Corina Russwurm ◽

...

Keyword(s):

Energy Transfer ◽

Fluorescence Resonance Energy Transfer ◽

Systematic Approach ◽

Dynamic Range ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Living Cells ◽

Binding Domains ◽

Fluorescence Resonance

The intracellular signalling molecule cGMP regulates a variety of physiological processes, and so the ability to monitor cGMP dynamics in living cells is highly desirable. Here, we report a systematic approach to create FRET (fluorescence resonance energy transfer)-based cGMP indicators from two known types of cGMP-binding domains which are found in cGMP-dependent protein kinase and phosphodiesterase 5, cNMP-BD [cyclic nucleotide monophosphate-binding domain and GAF [cGMP-specific and -stimulated phosphodiesterases, Anabaena adenylate cyclases and Escherichia coli FhlA] respectively. Interestingly, only cGMP-binding domains arranged in tandem configuration as in their parent proteins were cGMP-responsive. However, the GAF-derived sensors were unable to be used to study cGMP dynamics because of slow response kinetics to cGMP. Out of 24 cGMP-responsive constructs derived from cNMP-BDs, three were selected to cover a range of cGMP affinities with an EC50 between 500 nM and 6 μM. These indicators possess excellent specifity for cGMP, fast binding kinetics and twice the dynamic range of existing cGMP sensors. The in vivo performance of these new indicators is demonstrated in living cells and validated by comparison with cGMP dynamics as measured by radioimmunoassays.

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Functional complementation of high-efficiency resonance energy transfer: a new tool for the study of protein binding interactions in living cells

Biochemical Journal ◽

10.1042/bj20070803 ◽

2007 ◽

Vol 409 (1) ◽

pp. 251-261 ◽

Cited By ~ 37

Author(s):

Paola Molinari ◽

Ida Casella ◽

Tommaso Costa

Keyword(s):

Energy Transfer ◽

Mammalian Cells ◽

High Efficiency ◽

Dynamic Range ◽

Resonance Energy Transfer ◽

Light Output ◽

Resonance Energy ◽

Functional Complementation ◽

Single Chain ◽

Native Proteins

Green bioluminescence in Renilla species is generated by a ∼100% efficient RET (resonance energy transfer) process that is caused by the direct association of a blue-emitting luciferase [Rluc (Renilla luciferase)] and an RGFP (Renilla green fluorescent protein). Despite the high efficiency, such a system has never been evaluated as a potential reporter of protein–protein interactions. To address the question, we compared and analysed in mammalian cells the bioluminescence of Rluc and RGFP co-expressed as free native proteins, or as fused single-chain polypeptides and tethered partners of self-assembling coiled coils. Here, we show that: (i) no spontaneous interactions generating detectable BRET (bioluminescence RET) signals occur between the free native proteins; (ii) high-efficiency BRET similar to that observed in Renilla occurs in both fusion proteins and self-interacting chimaeras, but only if the N-terminal of RGFP is free; (iii) the high-efficiency BRET interaction is associated with a dramatic increase in light output when the luminescent reaction is triggered by low-quantum yield coelenterazine analogues. Here, we propose a new functional complementation assay based on the detection of the high-efficiency BRET signal that is generated when the reporters Rluc and RGFP are brought into close proximity by a pair of interacting proteins to which they are linked. To demonstrate its performance, we implemented the assay to measure the interaction between GPCRs (G-protein-coupled receptors) and β-arrestins. We show that complementation-induced BRET allows detection of the GPCR–β-arrestin interaction in a simple luminometric assay with high signal-to-noise ratio, good dynamic range and rapid response.

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Paper-Based Solid-Phase Multiplexed Nucleic Acid Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As Donors in Fluorescence Resonance Energy Transfer

Analytical Chemistry ◽

10.1021/ac401471n ◽

2013 ◽

Vol 85 (15) ◽

pp. 7502-7511 ◽

Cited By ~ 53

Author(s):

M. Omair Noor ◽

Ulrich J. Krull

Keyword(s):

Quantum Dots ◽

Energy Transfer ◽

Nucleic Acid ◽

Fluorescence Resonance Energy Transfer ◽

Dynamic Range ◽

Solid Phase ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Nucleic Acid Hybridization ◽

Hybridization Assay

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Design of metalloporphyrin-based dendritic nanoprobes for two-photon microscopy of oxygen

Journal of Porphyrins and Phthalocyanines ◽

10.1142/s1088424608000649 ◽

2008 ◽

Vol 12 (12) ◽

pp. 1261-1269 ◽

Cited By ~ 44

Author(s):

Artem Y. Lebedev ◽

Thomas Troxler ◽

Sergei A. Vinogradov

Keyword(s):

Electron Transfer ◽

Energy Transfer ◽

Dynamic Range ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Triplet States ◽

Redox Potentials ◽

Oxygen Quenching ◽

Two Photon Microscopy ◽

Two Photon

Metalloporphyrin-based phosphorescent nanoprobes are being developed for two-photon microscopy of oxygen. In these molecular constructs, the generation of porphyrin triplet states following two-photon excitation is induced by the intramolecular Förster-type resonance energy transfer from a covalently attached 2P antenna. In the earlier developed prototypes, electron transfer between the antenna and the metalloporphyrin strongly interferred with the phosphorescence, reducing the sensitivity and the dynamic range of the sensors. By tuning the distances between the antenna and the core, and adjusting their redox potentials, the unwanted electron transfer could be prevented. An array of phosphorescent Pt porphyrins (energy transfer acceptors) and 2P dyes (energy transfer donors) was screened using dynamic quenching of phosphorescence, and the FRET-pair with the minimal ET rate was identified. This pair, consisting of Coumarin-343 and Pt meso-tetra-(4-alkoxyphenyl)porphyrin, was used to construct a probe in which the antenna fragments were linked to the termini of G3 poly(arylglycine) (AG) dendrimer with PtP core. The folded dendrimer formed an insulating layer between the porphyrin and the antenna, simultaneously controlling the rate of oxygen quenching (Stern-Volmer oxygen quenching constant). Modification of the dendrimer periphery with oligoethyleneglycol residues made the probe's signal insensitive to the presence of proteins and other macromolecular solutes.

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A Homogeneous Single-Label Time-Resolved Fluorescence cAMP Assay

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057110397356 ◽

2011 ◽

Vol 16 (3) ◽

pp. 356-362 ◽

Cited By ~ 10

Author(s):

Eija Martikkala ◽

Anita Rozwandowicz-Jansen ◽

Pekka Hänninen ◽

Ulla Petäjä-Repo ◽

Harri Härmä

Keyword(s):

Energy Transfer ◽

Adenylyl Cyclase ◽

High Throughput ◽

Dynamic Range ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Time Resolved Fluorescence ◽

Time Resolved ◽

Gpcr Activation ◽

Label Time

G-protein–coupled receptors (GPCRs) are an important class of pharmaceutical drug targets. Functional high-throughput GPCR assays are needed to test an increasing number of synthesized novel drug compounds and their function in signal transduction processes. Measurement of changes in the cyclic adenosine monophosphate (cAMP) concentration is a widely used method to verify GPCR activation in the adenylyl cyclase pathway. Here, a single-label time-resolved fluorescence and high-throughput screening (HTS)–feasible method was developed to measure changes in cAMP levels in HEK293i cells overexpressing either β2-adrenergic or δ-opioid receptors. In the quenching resonance energy transfer (QRET) technique, soluble quenchers reduce the signal of unbound europium(III)-labeled cAMP in solution, whereas the antibody-bound fraction is fluorescent. The feasibility of this homogeneous competitive assay was proven by agonist-mediated stimulation of receptors coupled to either the stimulatory Gs or inhibitory Gi proteins. The reproducibility of the assays was excellent, and Z′ values exceeded 0.7. The dynamic range, signal-to-background ratio, and detection limit were compared with a commercial time-resolved fluorescence resonance energy transfer (TR-FRET) assay. In both homogeneous assays, similar assay parameters were obtained when adenylyl cyclase was stimulated directly by forskolin or via agonist-mediated activation of the Gs-coupled β2AR. The advantage of using the single-label approach relates to the cost-effectiveness of the QRET system compared with the two-label TR-FRET assay as there is no need for labeling of two binding partners leading to reduced requirements for assay optimization.

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Engineering with NanoLuc: a playground for the development of bioluminescent protein switches and sensors

Biochemical Society Transactions ◽

10.1042/bst20200440 ◽

2020 ◽

Vol 48 (6) ◽

pp. 2643-2655

Author(s):

Lieuwe Biewenga ◽

Bas J.H.M. Rosier ◽

Maarten Merkx

Keyword(s):

Energy Transfer ◽

Cell Biology ◽

Chemical Biology ◽

Dynamic Range ◽

State Of The Art ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Bioluminescence Resonance Energy Transfer ◽

Advantages And Disadvantages ◽

Bioluminescent Protein

The small engineered luciferase NanoLuc has rapidly become a powerful tool in the fields of biochemistry, chemical biology, and cell biology due to its exceptional brightness and stability. The continuously expanding NanoLuc toolbox has been employed in applications ranging from biosensors to molecular and cellular imaging, and currently includes split complementation variants, engineering techniques for spectral tuning, and bioluminescence resonance energy transfer-based concepts. In this review, we provide an overview of state-of-the-art NanoLuc-based sensors and switches with a focus on the underlying protein engineering approaches. We discuss the advantages and disadvantages of various strategies with respect to sensor sensitivity, modularity, and dynamic range of the sensor and provide a perspective on future strategies and applications.

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Design and Application of Highly Responsive Fluorescence Resonance Energy Transfer Biosensors for Detection of Sugar in Living Saccharomyces cerevisiae Cells

Applied and Environmental Microbiology ◽

10.1128/aem.01080-07 ◽

2007 ◽

Vol 73 (22) ◽

pp. 7408-7414 ◽

Cited By ~ 37

Author(s):

Jae-Seok Ha ◽

Jae Jun Song ◽

Young-Mi Lee ◽

Su-Jin Kim ◽

Jung-Hoon Sohn ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Energy Transfer ◽

Fluorescence Resonance Energy Transfer ◽

Signal Intensity ◽

Binding Proteins ◽

Dynamic Range ◽

Binding Protein ◽

Resonance Energy Transfer ◽

Resonance Energy

ABSTRACT A protein sensor with a highly responsive fluorescence resonance energy transfer (FRET) signal for sensing sugars in living Saccharomyces cerevisiae cells was developed by combinatorial engineering of the domain linker and the binding protein moiety. Although FRET sensors based on microbial binding proteins have previously been created for visualizing various sugars in vivo, such sensors are limited due to a weak signal intensity and a narrow dynamic range. In the present study, the length and composition of the linker moiety of a FRET-based sensor consisting of CFP-linker1-maltose-binding protein-linker2-YFP were redesigned, which resulted in a 10-fold-higher signal intensity. Molecular modeling of the composite linker moieties, including the connecting peptide and terminal regions of the flanking proteins, suggested that an ordered helical structure was preferable for tighter coupling of the conformational change of the binding proteins to the FRET response. When the binding site residue Trp62 of the maltose-binding protein was diversified by saturation mutagenesis, the Leu mutant exhibited an increased binding constant (82 μM) accompanied by further improvement in the signal intensity. Finally, the maltose sensor with optimized linkers was redesigned to create a sugar sensor with a new specificity and a wide dynamic range. When the optimized maltose sensors were employed as in vivo sensors, highly responsive FRET images were generated from real-time analysis of maltose uptake of Saccharomyces cerevisiae (baker's yeast).

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An enhanced molecular tension sensor based on bioluminescence resonance energy transfer (BRET)

10.1101/617696 ◽

2019 ◽

Author(s):

Eric J. Aird ◽

Kassidy J. Tompkins ◽

Wendy R. Gordon

Keyword(s):

Energy Transfer ◽

Dynamic Range ◽

Resonance Energy Transfer ◽

Focal Adhesions ◽

Resonance Energy ◽

Cellular Protein ◽

Bioluminescence Resonance Energy Transfer ◽

Tension Sensor ◽

Focal Adhesion Protein

ABSTRACTMolecular tension sensors measure piconewton forces experienced by individual proteins in the context of the cellular microenvironment. Current genetically-encoded tension sensors use FRET to report on extension of an elastic peptide encoded in a cellular protein of interest. Here we present the development and characterization of a new type of molecular tension sensor based on bioluminescence resonance energy transfer (BRET) which exhibits more desirable spectral properties and an enhanced dynamic range compared to other molecular tension sensors. Moreover, it avoids many disadvantages of FRET measurements in cells, including heating of the sample, autofluorescence, photobleaching, and corrections of direct acceptor excitation. We benchmark the sensor by inserting it into the canonical mechanosensing focal adhesion protein vinculin, observing highly resolved gradients of tensional changes across focal adhesions. We anticipate that the BRET-TS will expand the toolkit available to study mechanotransduction at a molecular level and allow potential extension to an in vivo context.

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Gated Resonance Energy Transfer (gRET) Controlled by Programmed Death Protein Ligand 1

Nanomaterials ◽

10.3390/nano10081592 ◽

2020 ◽

Vol 10 (8) ◽

pp. 1592 ◽

Cited By ~ 1

Author(s):

Hubert Grel ◽

Katarzyna Ratajczak ◽

Slawomir Jakiela ◽

Magdalena Stobiecka

Keyword(s):

Energy Transfer ◽

Dynamic Range ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Pharmaceutical Formulations ◽

Supramolecular Interactions ◽

Probe Molecule ◽

Checkpoint Control ◽

Plasmonic Nanoparticle ◽

Programmed Death

The resonance energy transfer (RET) between an excited fluorescent probe molecule and a plasmonic nanoparticle (AuNP) has been investigated to evaluate the effect of protein molecules on the RET efficiency. We have found that the energy transfer to a functionalized AuNP can be modulated by a sub-monolayer film of programmed death-ligand 1 (PD-L1) protein. The interactions of PD-L1 with AuNP@Cit involve incorporation of the protein in AuNP shell and formation of a submonolayer adsorption film with voids enabling gated surface plasmon resonance energy transfer (SPRET). A model of the gated-RET system based on the protein size, estimated using Fisher–Polikarpov–Craievich density approximation, has been developed and can be utilized for other proteins, with minimum data requirement, as well. The value of the equilibrium constant KL determined for the Langmuir isotherm is high: KL = 1.27 × 108 M−1, enabling highly sensitive control of the gated-RET by PD-L1. Thus, with the gated-RET technique, one can determine PD-L1 within the dynamic range, extending from 1.2 to 50 nM. Moreover, we have found that the Gibbs free energy for PD-L1 binding to AuNP@Cit is −46.26 kJ/mol (−11.05 kcal/mol), indicating a strong adsorption with supramolecular interactions. The proposed gated-RET system, with the fluorescence intensity of the fluorophore probe molecule modulated by plasmonic quenching with AuNP and shielding of energy transfer by the adsorbed PD-L1 can be further developed for determination of PD-L1 in pharmaceutical formulations for immune checkpoint control in cancer therapy.

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