This study presents a detailed procedure to perform single-molecule fluorescence resonance energy transfer (smFRET) experiments on G protein-coupled receptors (GPCRs) using site-specific labeling via unnatural amino acid (UAA) incorporation. The protocol provides a step-by-step guide for smFRET sample preparation, experiments, and data analysis.
The ability of cells to respond to external signals is essential for cellular development, growth, and survival. To respond to a signal from the environment, a cell must be able to recognize and process it. This task mainly relies on the function of membrane receptors, whose role is to convert signals into the biochemical language of the cell. G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptor proteins in humans. Among GPCRs, metabotropic glutamate receptors (mGluRs) are a unique subclass that function as obligate dimers and possess a large extracellular domain that contains the ligand-binding site. Recent advances in structural studies of mGluRs have improved the understanding of their activation process. However, the propagation of large-scale conformational changes through mGluRs during activation and modulation is poorly understood. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful technique to visualize and quantify the structural dynamics of biomolecules at the single-protein level. To visualize the dynamic process of mGluR2 activation, fluorescent conformational sensors based on unnatural amino acid (UAA) incorporation were developed that allowed site-specific protein labeling without perturbation of the native structure of receptors. The protocol described here explains how to perform these experiments, including the novel UAA labeling approach, sample preparation, and smFRET data acquisition and analysis. These strategies are generalizable and can be extended to investigate the conformational dynamics of a variety of membrane proteins.
The transfer of information across the plasma membrane is heavily dependent on the function of membrane receptors1. Ligand binding to a receptor leads to a conformational change and receptor activation. This process is often allosteric in nature2. With over 800 members, G protein-coupled receptors (GPCRs) are the largest family of membrane receptors in humans3. Due to their role in nearly all cellular processes, GPCRs have become important targets for therapeutic development. In the canonical model of GPCR signaling, agonist activation results in conformational changes of the receptor that subsequently activate the heterotrimeric G protein complex via exchange of GDP for GTP at the nucleotide binding pocket of Gα. The activated Gα-GTP and Gβγ subunits then control the activity of downstream effector proteins and propagate the signaling cascade4,5. This signaling process essentially depends on the ability of ligands to change the three-dimensional shape of the receptor. A mechanistic understanding of how ligands achieve this is critical for developing new therapeutics and designing synthetic receptors and sensors.
Metabotropic glutamate receptors (mGluRs) are members of the class C GPCR family and are important for the slow neuromodulatory effects of glutamate and tuning neuronal excitability6,7. Among all GPCRs, class C GPCRs are structurally unique in that they function as obligate dimers. mGluRs contain three structural domains: the Venus flytrap (VFT) domain, cysteine-rich domain (CRD), and transmembrane domain (TMD)8. The conformational changes during the activation process are complex and involve local and global conformational coupling that propagate over a 12 nm distance, as well as dimer cooperativity. The intermediate conformations, temporal ordering of states, and rate of transition between states are unknown. By following the conformation of individual receptors in real time, it is possible to identify the transient intermediate states and the sequence of conformational changes during activation. This can be achieved by applying single-molecule fluorescence resonance energy transfer9,10 (smFRET), as was recently applied to visualize the propagation of conformational changes during the activation of mGluR211. A key step in FRET experiments is the generation of FRET sensors by site-specific insertion of the donor and acceptor fluorophores into the protein of interest. An unnatural amino acid (UAA) incorporation strategy was adopted12,13,14,15 to overcome the limitations of typical site-specific fluorescent labeling technologies that require the creation of cysteine-less mutants or the insertion of a large genetically encoded tag. This allowed the conformational rearrangement of the essential compact allosteric linker, which joined the ligand-binding and signaling domains of mGluR2, to be observed. In this protocol, a step-by-step guide to performing smFRET experiments on mGluR2 is presented, including the approach for site-specific labeling of mGluR2 with UAA to attach fluorophores using the copper-catalyzed azide cyclization reaction. Moreover, this protocol describes the methodology for the direct capture of membrane proteins and data analysis. The protocol outlined here is also applicable to studying the conformational dynamics of other membrane proteins.
The overall workflow of the protocol is described in Figure 1.
1. Preparation of the sample chamber
2. mGluR2 expression with incorporated unnatural amino acid, fluorescent labeling, and extraction
NOTE: This protocol outlines the preparation, reagents, and treatment of cells for expressing mGluR2 containing the UAA 4-azido-L-phenylalanine (AZP). The procedure is for HEK293T cells grown on 18 mm glass coverslips. The procedure can be scaled up as necessary.
3. Single-molecule flow chamber assembly and functionalization
4. Single-molecule buffers
5. Microscope setup and smFRET data acquisition
6. Data analysis
Expression and fluorescent labeling of UAA-based FRET sensor
Herein, exemplary results of the insertion and fluorescent labeling of a UAA (AZP) within the CRD of mGluR2 (548UAA) are discussed11. As mentioned previously, to insert AZP into mGluR2, co-expression of the engineered translational machinery, which includes a modified tRNA synthetase and complementary tRNA (pIRE4-Azi), and mGluR2 containing an amber codon at position 548, created using mutagenesis, is necessary (Figure 2A,B). The labeling of AZP by cyanine dyes is achieved by a copper-catalyzed cycloaddition reaction (Figure 2C) and results in effective plasma membrane labeling of 548UAA (Figure 2D). To verify the translation of full-length 548UAA and the integrity of the dimeric receptor, SDS-PAGE electrophoresis was performed on the cell lysate from HEK293T cells expressing 548UAA labeled with Cy5. A single band at 250KDa was observed, which coincided with the full-length dimeric mGluR2 (Figure 2E).
Data acquisition and analysis
Cells expressing 548UAA with C-terminal FLAG-tag were labeled with Cy3 (donor) and Cy5 (acceptor) and then lysed with detergent31 in the presence of a protease inhibitor for in vitro study. Upon the completion of cell lysis and removal of the insoluble fraction by centrifugation, the supernatant was applied onto a polyethylene glycol (PEG)-passivated coverslip functionalized with an anti-FLAG-tag antibody for total internal reflection fluorescence (TIRF) imaging (Figure 3). The sample was illuminated using a 532 nm laser, and particles were selected for downstream analysis using smCamera software. Raw donor, acceptor, and FRET traces were generated for all selected molecules in smCamera and selected using MATLAB (section 6; Figure 4). An 8.8% donor bleed-through correction was applied to acceptor intensities for the experimental setup used here. This correction factor will vary with the experimental setup, dichroic filters, and emission filters used and should be determined by measuring the donor and acceptor signals under standard experimental conditions using cells labeled with donor fluorophore only and calculating the bleed-through ([acceptor intensity]/[donor intensity]). Figure 4 shows several representative FRET traces and corresponding donor and acceptor signals. These traces were selected using criteria previously described in the protocol (section 6). Representative selected traces that show transitions between multiple states and donor and acceptor bleaching events are shown in Figure 4B–D.
Identification of conformational states
To identify the conformational states 548UAA occupied and the relationship of these states relative to one another, Hidden Markov modeling (HMM) analysis was conducted. HMM analysis was performed using the vbFRET program executed on MATLAB28 (Figure 5A). Figure 5 uses data from an intermediate glutamate concentration (5µM) to illustrate the process of state identification. Based on raw smFRET traces, it was hypothesized that up to four potential FRET states existed for the CRD. Thus, the number of states was constrained to one to four. Overall, 25 iterations of fitting were performed for each trace to determine the number of states present. From these idealized fits, transitions between discrete FRET states can be extracted and plotted as a transition density heatmap (Figure 5B). The heatmap highlights four discrete FRET states at 0.31, 0.51, 0.71, and 0.89, indicated by dotted lines. Transitions were defined as changes in FRET >0.1. The idealized FRET traces also yield information on dwell-time for each identified conformation (Figure 5C).
Population FRET histogram generation and peak fitting
Representative single-molecule traces for 548UAA in the presence of varying glutamate concentrations that were manually selected for further analysis are shown in Figure 6A,B. A general analysis for smFRET experiments is to generate population histograms from hundreds of smFRET traces for each experimental condition (Figure 6C). Population FRET histograms are created from the segment of traces before bleaching. To avoid biasing the histogram toward the behavior of longer traces, it is necessary to generate a normalized FRET histogram from each trace prior to averaging. This ensures each trace contributes equally to the final histogram. In this system, the FRET histograms show a general shift toward higher FRET with increasing glutamate concentration, indicating a reduction in the distance between the CRDs and a shift toward the active conformation. However, regardless of the glutamate concentration, the FRET signal remains quite sporadic, indicating a high degree of intrinsic dynamics for the CRD. In addition to the general shift toward higher FRET, a redistribution of the conformational ensemble becomes apparent in the histogram (color curves). Individual molecules can also be seen visiting these conformational states (dashed lines) (Figure 6B). To determine the probability of state occupancy, one must divide the area of the peak by the total area, defined as the sum of all four individual peak areas. The smFRET histogram generated from the smFRET experiment of the CRD of mGluR2 displayed four dynamic states with peaks at 0.31, 0.51, 0.71, and 0.89 (labeled at states 1-4), respectively.
Figure 1: Flow chart of the working protocol and data analysis. Please click here to view a larger version of this figure.
Figure 2: Site-specific labeling of mGluR2 by click chemistry. (A) Schematic of the unnatural amino acid 4-azido-L-phenylalanine incorporation process in cells. (B) Schematic showing site-specific fluorescent labeling of mGluR2, with the unnatural amino acid at position 548, by copper-catalyzed azide-alkyne click reaction. (C) Three-dimensional structure of fluorescently labeled mGluR2 (donor molecule in green, acceptor molecule in red) with Cy3 and Cy5 molecules docked. (D) Representative confocal microscope image of HEK293T cells expressing 548UAA with the cell surface population labeled with donor (green: Cy3) and acceptor (red: Cy5) fluorophores through click chemistry. Scale bars = 10 µm. (E) Image of non-reducing 4%-20% polyacrylamide gel electrophoresis of cell lysate from HEK293T cells expressing 548UAA and labeled by Cy5-alkyne. The gel is imaged with a 633 nm excitation wavelength and 670-BP30 emission filter-Lane a: protein ladder; lane b: cell lysate; lane c: Cy5-alkyne dye. Results are representative of an individual experiment. Panels B, D, and E are reused from Liauw et al.11. Please click here to view a larger version of this figure.
Figure 3: Schematic representation of the smFRET experiments and microscope setup (TIRF). The single-molecule fluorescence image of the donor is shown in green (scale bar = 1000 nm) and the acceptor in red. The donor was excited at 532 nm using a laser. The acceptor is excited by FRET from the donor. Please click here to view a larger version of this figure.
Figure 4: Intensity traces of donor (green) and acceptor (red) labeled mGluR2. (A) Cartoon showing the receptor dynamics and change in distance between FRET probes. (B) Long-lived high FRET state. (C) Multiple FRET states with acceptor photobleaching first followed by donor photobleaching. (D) Short-lived FRET state with acceptor photobleaching. (E) Long-lived stable FRET state with acceptor photobleaching. This figure is reproduced with minimal modification from Liauw et al.11. Please click here to view a larger version of this figure.
Figure 5: Conformational state identification. (A) Representative FRET trace with idealized fit overlaid (red) along with the corresponding donor and acceptor signal. (B) Transition density heatmap highlighting the most frequent conformational transitions undergone by 548UAA. Dashed lines indicate FRET states. (C) Average dwell-times of each conformational state. This figure is reproduced with a minimal modification from Liauw et al.11. Please click here to view a larger version of this figure.
Figure 6: Single-molecule FRET reveals four conformational states of mGluR2 CRD. (A) Representative frame from a single-molecule movie with the donor channel (Cy3) on the left and the acceptor channel (Cy5) on the right. Molecules selected by analysis software for downstream processing are indicated by green circles. Scale bar = 3 µm. (B) Example single-molecule time traces of the 548UAA at different glutamate concentrations. Donor (green) and acceptor (red) intensities and the corresponding FRET (blue) are shown. Dashed lines represent four distinct FRET states. (C) smFRET population histograms in a range of glutamate concentrations. Data represent mean ± SEM. of N = 3 independent experiments. This figure is reproduced with a minimal modification from Liauw et al.11. Please click here to view a larger version of this figure.
Figure 7: Summary of the overall protocol. (A) Workflow for growing and labeling cells expressing a protein containing an unnatural amino acid. (B) Single-molecule FRET experimental and analysis workflow used to identify conformational states and characterize the dynamic properties of the CRD domain in mGluR2. Please click here to view a larger version of this figure.
Component | Volume/Reaction (μL) |
Reduced Serum Medium | 100 |
Transfection reagent | 4.4 |
Component | Volume/Reaction (μL) |
Reduced Serum Medium | 100 |
P3000 | 4 |
Construct/Component Name | Concentration (ng/μL) | Volume/well (12-well) (μL) | Wells (#) | DNA Added (μL) |
tRNA/synthetase | 1000 | 1 | 1 | 1 |
Amber codon containing protein | 1000 | 1 | 1 | 1 |
Table 1: Reagents for the transfection of the HEK 293 T cell.
Reagents | Volume to add (µL) | Stock conc (mM) | Final Conc (mM) |
1x RB | 655.5 | ||
BTTES | 10.5 | 50 | 0.75 |
CuSO4 | 5.25 | 20 | 0.15 |
NaAsc | 17.5 | 100 | 2.5 |
AminoG | 8.75 | 100 | 1.25 |
Cy3-Alkyne (10 mM) | 1.25 | 10 | 0.018 |
Cy5-Alkyne (10 mM) | 1.25 | 10 | 0.018 |
Table 2: Composition of the labeling solution (click chemistry).
Supplementary File 1: Composition of various buffers used in this study. Please click here to download this File.
GPCRs are proteins that operate on the cell membrane to initiate signal transduction. Many GPCRs consist of multiple domains, with signaling being dependent on the cooperative interaction between the domains. To modulate the properties of these membrane receptors, it is essential to understand the dynamic behavior of the multiple domains. Single-molecule fluorescence resonance energy transfer (smFRET) is a fluorescence technique that enables the measurement of protein conformation and dynamics in real time11,32. Here, an approach combining smFRET, single-molecule pull-down (SiMPull), and total internal reflection fluorescence (TIRF) microscopy to directly visualize the rearrangement of individual proteins immobilized on a passivated surface is described5,11,33. FRET is highly sensitive to distance and effectively functions as a nanoscale ruler that is appropriate for probing intramolecular changes (3-8 nm). Compared to traditional biophysical approaches, smFRET is particularly well suited for the study of large conformational dynamics at a ~10 ms timescale and requires a small sample volume (approximately 1 fmole per experiment). Furthermore, in contrast to ensemble measurements of conformational dynamics, such as those provided by nuclear magnetic resonance (NMR)34 or double electron-electron resonance (DEER) spectroscopy35, smFRET allows the explicit assignment of both conformational states and their time ordering, as well as the direct detection of rare and transient intermediate states.
Currently, the limitations of site-specific fluorescent labeling pose a technical challenge that prevents the broader application of smFRET to study the conformation dynamics of proteins. Moreover, it is difficult to purify membrane proteins in large quantities and preserve their activity. Common labeling strategies utilize large protein tags or require the generation of minimal cysteine mutants, often restricting fluorophore conjugation to the termini of proteins with no exposed cysteine residues. To circumvent these limitations, an unnatural amino acid (UAA) incorporation strategy was adapted and optimized, allowing for non-perturbative, residue-specific labeling using a copper-catalyzed click reaction11,12,14. This strategy makes it possible to conjugate fluorophores throughout the solvent-exposed regions of the membrane receptor and enables a broader array of conformational sensors to be generated. In this protocol, a single type of conjugation chemistry is used. This results in donor-donor and acceptor-acceptor only populations, which are omitted during downstream analysis. Alternatively, two orthogonal labeling strategies can be used to avoid this or overcome possible heterogeneity when the protein of interest is not symmetric.
The direct capture of protein helps to bypass traditional purification steps that are time-consuming and technically challenging. Due to the cytotoxicity of copper, copper-free click chemistry based on trans-cyclooctane36 or methyl-tetrazine37 are also employed. However, those reagents are expensive, and the non-regiospecific38 nature of the reaction results in a low yield of fluorescently tagged protein of interest.
Thorough guidelines for in vitro smFRET experiments, starting from flow chamber preparation to data analysis, were presented here. The smFRET data were collected using a TIRF setup, and the analysis was performed using a custom-written MATLAB code.
A few key considerations should be noted. First, one should prepare the PEG passivated surface to be homogeneous and less dense with biotin-PEG. Excess biotin-PEG may result in excessive protein pull-down, making it difficult to resolve fluorescent signals from individual molecules. Second, the protein sample (cell lysate supernatant) should be thoroughly diluted before being added to the sample chamber to avoid surface saturation. The yield of protein depends on the cell density, detergent of choice, and detergent concentration. To optimize the number of single donor and acceptor pairs, one should target a density of ~400 molecules in a 256 x 512 pixel field of view. Third, the Trolox buffer should be stored at −20 °C for long-term usage (months). Trolox buffer remains stable for 2 weeks when stored at 4 °C. Lastly, care should be taken not to introduce air bubbles into the flow chamber after functionalization with the antibody. Drying of the flow chamber will result in reduced efficiency of protein immobilization and sample protein denaturation.
The conformational dynamics of the mGluR2 sensor described here were successfully examined at the individual receptor level using smFRET and provided insight into the mechanism of receptor activation. The CRD was found to demonstrate a high level of intrinsic dynamics, existing in an equilibrium among four conformational FRET states regardless of the presence or absence of glutamate. A shift toward higher FRET states or a more compact receptor was shown to occur in a glutamate concentration-dependent manner (Figure 6). Interestingly, even at saturating levels of glutamate, the CRD remained dynamic. The method presented here (summarized in Figure 7) to understand the conformational dynamics is applicable to other class C GPCRs, as well as other membrane proteins such as ion channels, ionotropic receptors, and receptor tyrosine kinases (RTKs)32,39.
The authors have nothing to disclose.
We thank members of the Reza Vafabakhsh lab for discussions. This work was supported by the National Institutes of Health grant R01GM140272 (to R.V.), by The Searle Leadership Fund for the Life Sciences at Northwestern University, and by the Chicago Biomedical Consortium with support from the Searle Funds at The Chicago Community Trust (to R.V.). B.W.L. was supported by the National Institute of General Medical Sciences (NIGMS) Training Grant T32GM-008061.
(+)-Sodium L-Ascorbate | Sigma Aldrich | Cat # 11140-250G | |
4-azido-L-phenylalanine | Chem-Impex International | Cat # 06162 | |
548UAA | Liauw et al. 2021 | Transfected construct | |
Acetic Acid | Fisher Chemical | 64-19-7 | |
Acetone | Fisher Chemical | 67-64-1 | |
Adobe Illustrator (2022) | https://www.adobe.com/ | RRID:SCR_010279 | Software, algorithm |
Aminoguanidine (hydrochloride) | Cayman Chemical | 81530 | |
Aminosilane | Aldrich | 919-30-2 | |
Bath Sonicator 2.8 L | Fisher Scientific | Ultrasonic Bath 2.8 L | |
Biotin-PEG | Laysan Bio Inc | Item# Biotin-PEG-SVA-5000-100mg | |
BTTES | Click Chemistry Tools | 1237-500 | |
Copper (II) sulfate | Sigma Aldrich | Cat # 451657-10G | |
Cover slip | VWR | 16004-306 | Sample chamber |
Cy3 Alkyne | Click Chemistry Tools | TA117-5 | |
Cy5 Alkyne | Click Chemistry Tools | TA116-5 | |
DDM | Anatrace | Part# D310 1 GM | Detergent |
DDM-CHS (10:1) | Anatrace | Part# D310-CH210 1 ML | Detergent with cholecterol |
Defined Fetal Bovine Serum | Thermo Fisher Scientific | SH30070.03 | |
Di01-R405/488/561/635 | Semrock | Notch filter | |
DMEM | Corning | 10-013-CV | |
EMCCD | Andor | DU-897U | Camera |
ET542lp | Chroma | Long pass emission filter | |
FF640-FDi01 | Semrock | Emission dichroic filter | |
FLAG-tag antibody | Genscript | A01429 | |
Fluorescent bead | Invitrogen T7279 | TetraSpeck microspheres | Spherical bead |
Glass slides | Fisherfinest | 12-544-4 | sample chamber |
Glutamate | Sigma Aldrich | Cat # 6106-04-3 | |
HEK 293T | Sigma Aldrich | Cat # 12022001 | Cell line |
HEPES | FisherBioReagents | 7365-45-9 | |
Image splitter | OptoSplit II | ||
KOH | Fluka | 1310-58-3 | |
Laser | Oxxius | 4-line laser combiner | |
Lipofectamine 3000 Transfection Reagent | Thermo Fisher Scientific | L3000015 | Transfection Reagent |
Methanol | Fisher Chemical | 67-56-1 | |
Microscope | Olympus | Olympus IX83 | |
Milli-Q water | Barnstead | Water Deionizer | |
m-PEG | Laysan Bio Inc | Item# MPEG-SIL-5000-1g | |
NF03-405/488/532/635 | Semrock | Dichroic mirror | |
OptiMEM | Thermo Fisher Scientific | 51985091 | Reduced Serum Medium |
OptiMEM/Reduced serum medium | Thermo Fisher Scientific | ||
OriginPro (2020b) | https://www.originlab.com/ | RRID:SCR_014212 | Data analysis and graphing software |
Penicillin-Streptomycin | Gibco | 15140-122 | |
pIRE4-Azi | Addgene | Plasmid # 105829 | Transfected construct |
Poly-L-lysine hydrobromide | Sigma Aldrich | Cat # P2636 | |
Protocatechuic acid (PCA) | HWI group | 99-50-3 | |
smCamera (Version 1.0) | http://ha.med.jhmi.edu/resources/ | Camera software | |
Sodium bicarbonate | FisherBioReagents | 144-55-8 | |
Sodium hydroxide (NaOH) | Sigma | 1310-73-2 | |
Syringe filter | Whatman UNIFLO | Cat#9914-2502 | Liquid filtration |
Trolox | Sigma | 53188-07 |