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.

<ol>
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The authors declare no competing interests.

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,&#160;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.&#160;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 &#8722;20 &#176;C for long-term usage (months). Trolox buffer remains stable for 2 weeks when stored at 4 &#176;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 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 G&#945;. The activated G&#945;-GTP and G&#946;&#947; 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.

<table><tbody><tr><td>(+)-Sodium L-Ascorbate</td><td>Sigma Aldrich</td><td>Cat # 11140-250G</td><td></td></tr><tr><td>4-azido-L-phenylalanine</td><td>Chem-Impex International</td><td>Cat # 06162</td><td></td></tr><tr><td>548UAA</td><td>Liauw et al. 2021</td><td></td><td>Transfected construct</td></tr><tr><td>Acetic Acid</td><td>Fisher Chemical</td><td>64-19-7</td><td></td></tr><tr><td>Acetone</td><td>Fisher Chemical</td><td>67-64-1</td><td></td></tr><tr><td>Adobe Illustrator (2022)</td><td>https://www.adobe.com/</td><td>RRID:SCR_010279</td><td>Software, algorithm</td></tr><tr><td>Aminoguanidine (hydrochloride)</td><td>Cayman Chemical</td><td>81530</td><td></td></tr><tr><td>Aminosilane</td><td>Aldrich</td><td>919-30-2</td><td></td></tr><tr><td>Bath Sonicator 2.8 L</td><td>Fisher Scientific</td><td></td><td>Ultrasonic Bath 2.8 L</td></tr><tr><td>Biotin-PEG</td><td>Laysan Bio Inc</td><td>Item# Biotin-PEG-SVA-5000-100mg</td><td></td></tr><tr><td>BTTES</td><td>Click Chemistry Tools</td><td>1237-500</td><td></td></tr><tr><td>Copper (II) sulfate</td><td>Sigma Aldrich</td><td>Cat # 451657-10G</td><td></td></tr><tr><td>Cover slip</td><td>VWR</td><td>16004-306</td><td>Sample chamber</td></tr><tr><td>Cy3 Alkyne</td><td>Click Chemistry Tools</td><td>TA117-5</td><td></td></tr><tr><td>Cy5 Alkyne</td><td>Click Chemistry Tools</td><td>TA116-5</td><td></td></tr><tr><td>DDM</td><td>Anatrace</td><td>Part# D310 1 GM</td><td>Detergent</td></tr><tr><td>DDM-CHS (10:1)</td><td>Anatrace</td><td>Part# D310-CH210 1 ML</td><td>Detergent with cholecterol</td></tr><tr><td>Defined Fetal Bovine Serum</td><td>Thermo Fisher Scientific</td><td>SH30070.03</td><td></td></tr><tr><td>Di01-R405/488/561/635</td><td>Semrock</td><td></td><td>Notch filter</td></tr><tr><td>DMEM</td><td>Corning</td><td>10-013-CV</td><td></td></tr><tr><td>EMCCD</td><td>Andor</td><td>DU-897U</td><td>Camera</td></tr><tr><td>ET542lp</td><td>Chroma</td><td></td><td>Long pass emission filter</td></tr><tr><td>FF640-FDi01</td><td>Semrock</td><td></td><td>Emission dichroic filter</td></tr><tr><td>FLAG-tag antibody</td><td>Genscript</td><td>A01429</td><td></td></tr><tr><td>Fluorescent bead</td><td>Invitrogen T7279</td><td>TetraSpeck microspheres</td><td>Spherical bead</td></tr><tr><td>Glass slides</td><td>Fisherfinest</td><td>12-544-4</td><td>sample chamber</td></tr><tr><td>Glutamate</td><td>Sigma Aldrich</td><td>Cat # 6106-04-3</td><td></td></tr><tr><td>HEK 293T</td><td>Sigma Aldrich</td><td>Cat # 12022001</td><td>Cell line</td></tr><tr><td>HEPES</td><td>FisherBioReagents</td><td>7365-45-9</td><td></td></tr><tr><td>Image splitter</td><td>OptoSplit II</td><td></td><td></td></tr><tr><td>KOH</td><td>Fluka</td><td>1310-58-3</td><td></td></tr><tr><td>Laser</td><td>Oxxius</td><td></td><td>4-line laser combiner</td></tr><tr><td>Lipofectamine 3000 Transfection Reagent</td><td>Thermo Fisher Scientific</td><td>L3000015</td><td>Transfection Reagent</td></tr><tr><td>Methanol</td><td>Fisher Chemical</td><td>67-56-1</td><td></td></tr><tr><td>Microscope</td><td>Olympus</td><td>Olympus IX83</td><td></td></tr><tr><td>Milli-Q water</td><td>Barnstead</td><td></td><td>Water Deionizer</td></tr><tr><td>m-PEG</td><td>Laysan Bio Inc</td><td>Item# MPEG-SIL-5000-1g</td><td></td></tr><tr><td>NF03-405/488/532/635</td><td>Semrock</td><td></td><td>Dichroic mirror</td></tr><tr><td>OptiMEM</td><td>Thermo Fisher Scientific</td><td>51985091</td><td>Reduced Serum Medium</td></tr><tr><td>OptiMEM/Reduced serum medium</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr><tr><td>OriginPro (2020b)</td><td>https://www.originlab.com/</td><td>RRID:SCR_014212</td><td>Data analysis and graphing software</td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>pIRE4-Azi</td><td>Addgene</td><td>Plasmid # 105829</td><td>Transfected construct</td></tr><tr><td>Poly-L-lysine hydrobromide</td><td>Sigma Aldrich</td><td>Cat # P2636</td><td></td></tr><tr><td>Protocatechuic acid (PCA)</td><td>HWI group</td><td>99-50-3</td><td></td></tr><tr><td>smCamera (Version 1.0)</td><td>http://ha.med.jhmi.edu/resources/</td><td></td><td>Camera software</td></tr><tr><td>Sodium bicarbonate</td><td>FisherBioReagents</td><td>144-55-8</td><td></td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>Sigma</td><td>1310-73-2</td><td></td></tr><tr><td>Syringe filter</td><td>Whatman UNIFLO</td><td>Cat#9914-2502</td><td>Liquid filtration</td></tr><tr><td>Trolox</td><td>Sigma</td><td>53188-07</td><td></td></tr></tbody></table>

visualizing the conformational dynamics of membrane receptors using single-molecule fret

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.

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&#160;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]).&#160;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 (5&#181;M) 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 &#62;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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64254/64254fig01.jpg" /> 
Figure 1: Flow chart of the working protocol and data analysis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64254/64254fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64254/64254fig02.jpg" /> 
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 &#181;m. (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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64254/64254fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64254/64254fig03.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64254/64254fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64254/64254fig04.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64254/64254fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64254/64254fig05.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64254/64254fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64254/64254fig06.jpg" /> 
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 &#181;m. (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 &#177; SEM. of N = 3 independent experiments. This figure is reproduced with a minimal modification from Liauw et al.11. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64254/64254fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/64254/64254fig07.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64254/64254fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Component</td>
			<td>Volume/Reaction (&#956;L)</td>
		</tr>
		<tr>
			<td>Reduced Serum Medium</td>
			<td>100</td>
		</tr>
		<tr>
			<td>Transfection reagent</td>
			<td>4.4</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Volume/Reaction (&#956;L)</td>
		</tr>
		<tr>
			<td>Reduced Serum Medium</td>
			<td>100</td>
		</tr>
		<tr>
			<td>P3000</td>
			<td>4</td>
		</tr>
	</tbody>
</table>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Construct/Component Name</td>
			<td>Concentration (ng/&#956;L)</td>
			<td>Volume/well (12-well) (&#956;L)</td>
			<td>Wells (#)</td>
			<td>DNA Added (&#956;L)</td>
		</tr>
		<tr>
			<td>tRNA/synthetase</td>
			<td>1000</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Amber codon containing protein</td>
			<td>1000</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
		</tr>
	</tbody>
</table>
Table 1: Reagents for the transfection of the HEK 293 T cell.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td>Volume to add (&#181;L)</td>
			<td>Stock conc (mM)</td>
			<td>Final Conc (mM)</td>
		</tr>
		<tr>
			<td>1x RB</td>
			<td>655.5</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BTTES</td>
			<td>10.5</td>
			<td>50</td>
			<td>0.75</td>
		</tr>
		<tr>
			<td>CuSO4</td>
			<td>5.25</td>
			<td>20</td>
			<td>0.15</td>
		</tr>
		<tr>
			<td>NaAsc</td>
			<td>17.5</td>
			<td>100</td>
			<td>2.5</td>
		</tr>
		<tr>
			<td>AminoG</td>
			<td>8.75</td>
			<td>100</td>
			<td>1.25</td>
		</tr>
		<tr>
			<td>Cy3-Alkyne (10 mM)</td>
			<td>1.25</td>
			<td>10</td>
			<td>0.018</td>
		</tr>
		<tr>
			<td>Cy5-Alkyne (10 mM)</td>
			<td>1.25</td>
			<td>10</td>
			<td>0.018</td>
		</tr>
	</tbody>
</table>
Table 2: Composition of the labeling solution (click chemistry).
Supplementary File 1: Composition of various buffers used in this study. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64254/Supplementary File 1_64254R1.docx" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Visualizing the Conformational Dynamics of Membrane Receptors Using Single-Molecule FRET at JoVE.com

Visualizing the Conformational Dynamics of Membrane Receptors Using Single-Molecule FRET

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 ...

visualizing-conformational-dynamics-membrane-receptors-using-single

Nanyang Technological University

Research

JoVE Journal

Biochemistry

3.1K Views.  Northwestern University. Hier gebruikten we fret met één molecuul en sitespecifieke eiwitetikettering via een natuurlijke aminozuurincorporatie om de conformatiedynamiek van mGluR2 te bestuderen. Dit maakt het mogelijk om de eiwitdynamiek te bestuderen zonder de eiwitstructuur te verstoren. Atomaire structuur geeft een statisch beeld van een eiwit.Single-molecule FRET stelt ons echter in staat om het volledige bereik van eiwitbewegingen in realtime en oplossing te visualiseren. Deze methode kan worden toegep...

Video: Visualizing the Conformational Dynamics of Membrane Receptors Using Single-Molecule FRET

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all &#34;dimensions&#34; of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 &#197; in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 &#197; for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

A protocol for high-precision FRET experiments at the single molecule level is presented here. Additionally, this methodology can be used to identify three conformational states in the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor. Determining precise distances is the first step towards building structural models based on FRET experiments.

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

Bioengineering

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions are likely affected by the mechanical environment in which binding and signaling take place. A recent study demonstrated that mechanical force can regulate antigen recognition by and triggering of the T-cell receptor (TCR). This was made possible by a new technology we developed and termed fluorescence biomembrane force probe (fBFP), which combines single-molecule force spectroscopy with fluorescence microscopy. Using an ultra-soft human red blood cell as the sensitive force sensor, a high-speed camera and real-time imaging tracking techniques, the fBFP is of ~1 pN (10-12 N), ~3 nm and ~0.5 msec in force, spatial and temporal resolution. With the fBFP, one can precisely measure single receptor-ligand binding kinetics under force regulation and simultaneously image binding-triggered intracellular calcium signaling on a single live cell. This new technology can be used to study other membrane receptor-ligand interaction and signaling in other cells under mechanical regulation.

We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions ...

Single-Molecule Imaging of Lateral Mobility and Ion Channel Activity in Lipid Bilayers using Total Internal Reflection Fluorescence (TIRF) Microscopy

High-resolution imaging techniques have shown that many ion channels are not static, but subject to highly dynamic processes, including the transient association of pore-forming and auxiliary subunits, lateral diffusion, and clustering with other proteins. However, the relationship between lateral diffusion and function is poorly understood. To approach this problem, we describe how lateral mobility and activity of individual channels in supported lipid membranes can be monitored and correlated using total internal reflection fluorescence (TIRF) microscopy. Membranes are fabricated on an ultrathin hydrogel substrate using the droplet interface bilayer (DIB) technique. Compared to other types of model membranes, these membranes have the advantage of being mechanically robust and suitable for highly sensitive analytical techniques. This protocol measures Ca2+ ion flux through single channels by observing the fluorescence emission of a Ca2+-sensitive dye in close proximity to the membrane. In contrast to classical single-molecule tracking approaches, no fluorescent fusion proteins or labels, which can interfere with lateral movement and function in the membrane, are required. Possible changes in ion flux associated with conformational changes of the protein are only due to protein lateral motion in the membrane. Representative results are shown using the mitochondrial protein translocation channel TOM-CC and the bacterial channel OmpF. In contrast to OmpF, the gating of TOM-CC is very sensitive to molecular confinement and the nature of lateral diffusion. Hence, supported droplet-interface bilayers are a powerful tool to characterize the link between lateral diffusion and the function of ion channels.

Single-Molecule Imaging of Lateral Mobility and Ion Channel Activity in Lipid Bilayers using Total Internal Reflection Fluorescence TIRF Microscopy

This protocol describes how to use TIRF microscopy to track individual ion channels and determine their activity in supported lipid membranes, thereby defining the interplay between lateral membrane movement and channel function. It describes how to prepare the membranes, record the data, and analyze the results.

High-resolution imaging techniques have shown that many ion channels are not static, but subject to highly dynamic processes, including the transient ...

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

Biology

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called mechanotransduction. A crucial step in mechanotransduction is the transmission of forces between the external and internal environments. To transmit forces, there must be a sustained, unbroken physical linkage created by a series of protein-protein interactions. For a given protein-protein interaction, mechanical load can either have no effect on the interaction, lead to faster disassociation of the interaction, or even stabilize the interaction. Understanding how molecular load dictates protein turnover in living cells can provide valuable information about the mechanical state of a protein, in turn elucidating its role in mechanotransduction. Existing techniques for measuring force-sensitive protein dynamics either lack direct measurements of protein load or rely on the measurements performed outside of the cellular context. Here, we describe a protocol for the F&#246;rster resonance energy transfer-fluorescence recovery after photobleaching (FRET-FRAP) technique, which enables the measurement of force-sensitive protein dynamics within living cells. This technique is potentially applicable to any FRET-based tension sensor, facilitating the study of force-sensitive protein dynamics in variety of subcellular structures and in different cell types.

Here, we present a protocol for the simultaneous use of F&#246;rster resonance energy transfer-based tension sensors to measure protein load and fluorescence recovery after photobleaching to measure protein dynamics enabling the measurement of force-sensitive protein dynamics within living cells.

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called ...

Monitoring GTPase Atlastin in Different Nucleotide Loading States

Single-Molecule FRET Imaging for Observing the Conformational Dynamics of Dynamin-Like GTPase Atlastin

The rigid-body rotation of the three-helical middle domain (3HB) relative to the GTPase domain of dynamin-like protein atlastin (ATL) is a crucial driver of homotypic membrane fusion within the endoplasmic reticulum (ER). Disruptions in this process have been associated with hereditary spastic paraplegia (HSP), a neurodegenerative disorder. Structural and biochemical studies suggest that the conformational changes in ATL are linked to GTP hydrolysis, but real-time visualization of these conformational dynamics during the GTP hydrolysis cycle remains challenging. To better understand the mechanical mechanisms behind ATL function, single-molecule F&#246;rster resonance energy transfer (smFRET) was utilized. Three specific strategies were employed to immobilize the N-terminal cytosolic region of human ATL1 (ATL1cyto) in a streptavidin-coated microfluidic chamber, facilitating the application of intramolecular and intermolecular smFRET imaging. This allowed precise monitoring of protein conformations in various nucleotide-loading states, providing direct insights into individual molecular behaviors. This method can be applied to study other mechanochemical proteins as well.

The functions of dynamin superfamily proteins depend on conformational changes coupled with GTP hydrolysis. A system is described using the single-molecule FRET (smFRET) technique to monitor the conformational dynamics of dynamin-like GTPase atlastin in different nucleotide-loading states.

The rigid-body rotation of the three-helical middle domain (3HB) relative to the GTPase domain of dynamin-like protein atlastin (ATL) is a crucial driver ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Automated Two-dimensional Spatiotemporal Analysis of Mobile Single-molecule FRET Probes

Single-molecule F&#246;rster resonance energy transfer (smFRET) is a versatile technique reporting on distances in the sub-nanometer to nanometer range. It has been used in a wide range of biophysical and molecular biological experiments, including the measurement of molecular forces, characterization of conformational dynamics of biomolecules, observation of intracellular colocalization of proteins, and determination of receptor-ligand interaction times. In a widefield microscopy configuration, experiments are typically performed using surface-immobilized probes. Here, a method combining single-molecule tracking with alternating excitation (ALEX) smFRET experiments is presented, permitting the acquisition of smFRET time traces of surface-bound, yet mobile probes in plasma membranes or glass-supported lipid bilayers. For the analysis of recorded data, an automated, open-source software collection was developed supporting (i) the localization of fluorescent signals, (ii) single-particle tracking, (iii) determination of FRET-related quantities including correction factors, (iv) stringent verification of smFRET traces, and (v) intuitive presentation of the results. The generated data can conveniently be used as input for further exploration via specialized software, e.g., for the assessment of the diffusional behavior of probes or the investigation of FRET transitions.

This article presents a method for spatiotemporal analysis of mobile, single-molecule F&#246;rster resonance energy transfer (smFRET)-based probes using widefield fluorescence microscopy. The newly developed software toolkit allows the determination of smFRET time traces of moving probes, including the correct FRET efficiency and the molecular positions, as functions of time.

Single-molecule F&#246;rster resonance energy transfer (smFRET) is a versatile technique reporting on distances in the sub-nanometer to nanometer range. ...

Visualizing the Conformational Dynamics of Membrane Receptors Using Single-Molecule FRET

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