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.

The overall workflow of the protocol is described in Figure 1.
1. Preparation of the sample chamber
<ol>
	<li>Slide and coverslip cleaning 
	NOTE: These steps aim to clean the surfaces of the slides as well as the coverslips and prepare them for aminosilanization. One critical requirement for conducting single-molecule fluorescence experiments on surface-tethered molecules is a passivated surface. The most reliable and reproducible passivation technique...

<ol>
	<li>Smock, R. G., Gierasch, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sending+signals+dynamically.">Sending signals dynamically.</a> Science. 324 (5924), 198-203 (2009).</li><li>Changeux, J. P., Christopoulos, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allosteric+modulation+as+a+unifying+mechanism+for+receptor+function+and+regulation.">Allosteric modulation as a unifying mechanism for receptor function and regulation.</a> Cell. 166 (5), 1084-1102 (2016).</li><li>Tang, X. -. l., Wang, Y., Li, D. -. l., Luo, J., Liu, M. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orphan+G+protein-coupled+receptors+(GPCRs):+biological+functions+and+potential+drug+targets.">Orphan G protein-coupled receptors (GPCRs): biological functions and potential drug targets.</a> Acta Pharmacologica Sinica. 33 (3), 363-371 (2012).</li><li>Chung, K. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+changes+in+the+G+protein+Gs+induced+by+the+&#946;2+adrenergic+receptor.">Conformational changes in the G protein Gs induced by the &#946;2 adrenergic receptor.</a> Nature. 477 (7366), 611-615 (2011).</li><li>Vafabakhsh, R., Levitz, J., Isacoff, E. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+dynamics+of+a+class+C+G-protein-coupled+receptor.">Conformational dynamics of a class C G-protein-coupled receptor.</a> Nature. 524 (7566), 497-501 (2015).</li><li>Niswender, C. M., Conn, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabotropic+glutamate+receptors:+Physiology,+pharmacology,+and+disease.">Metabotropic glutamate receptors: Physiology, pharmacology, and disease.</a> Annual Review of Pharmacology and Toxicology. 50, 295-322 (2010).</li><li>Pin, J. P., Bettler, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+and+functions+of+mGlu+and+GABA(B)+receptor+complexes.">Organization and functions of mGlu and GABA(B) receptor complexes.</a> Nature. 540 (7631), 60-68 (2016).</li><li>Kniazeff, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Closed+state+of+both+binding+domains+of+homodimeric+mGlu+receptors+is+required+for+full+activity.">Closed state of both binding domains of homodimeric mGlu receptors is required for full activity.</a> Nature Structural &amp; Molecular Biology. 11 (8), 706-713 (2004).</li><li>Ha, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+fluorescence+resonance+energy+transfer.">Single-molecule fluorescence resonance energy transfer.</a> Methods. 25 (1), 78-86 (2001).</li><li>Schuler, B., Eaton, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+folding+studied+by+single-molecule+FRET.">Protein folding studied by single-molecule FRET.</a> Current Opinion in Structural Biology. 18 (1), 16-26 (2008).</li><li>Liauw, B. W. -. H., Afsari, H. S., Vafabakhsh, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+rearrangement+during+activation+of+a+metabotropic+glutamate+receptor.">Conformational rearrangement during activation of a metabotropic glutamate receptor.</a> Nature Chemical Biology. 17 (3), 291-297 (2021).</li><li>Noren, C. J., Anthonycahill, S. J., Griffith, M. C., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+general+method+for+site-specific+incorporation+of+unnatural+amino+acids+into+proteins.">A general method for site-specific incorporation of unnatural amino acids into proteins.</a> Science. 244 (4901), 182-188 (1989).</li><li>Presolski, S. I., Hong, V. P., Finn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Copper-catalyzed+azide-alkyne+click+chemistry+for+bioconjugation.">Copper-catalyzed azide-alkyne click chemistry for bioconjugation.</a> Current Protocols in Chemical Biology. 3 (4), 153-162 (2011).</li><li>Huber, T., Naganathan, S., Tian, H., Ye, S. X., Sakmar, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unnatural+amino+acid+mutagenesis+of+GPCRs+using+amber+codon+suppression+and+bioorthogonal+labeling.">Unnatural amino acid mutagenesis of GPCRs using amber codon suppression and bioorthogonal labeling.</a> G Protein Coupled Receptors: Structure. 520, 281-305 (2013).</li><li>Serfling, R., Coin, I., Pecoraro, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+Four+-+Incorporation+of+Unnatural+Amino+Acids+into+Proteins+Expressed+in+Mammalian+Cells.">Chapter Four - Incorporation of Unnatural Amino Acids into Proteins Expressed in Mammalian Cells.</a> Methods in Enzymology. 580, 89-107 (2016).</li><li>Chandradoss, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+passivation+for+single-molecule+protein+studies.">Surface passivation for single-molecule protein studies.</a> Journal of Visualized Experiments. (86), e50549 (2014).</li><li>Rasnik, I., McKinney, S. A., Ha, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonblinking+and+long-lasting+single-molecule+fluorescence+imaging.">Nonblinking and long-lasting single-molecule fluorescence imaging.</a> Nature Methods. 3 (11), 891-893 (2006).</li><li>Cordes, T., Vogelsang, J., Tinnefeld, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+mechanism+of+Trolox+as+antiblinking+and+antibleaching+reagent.">On the mechanism of Trolox as antiblinking and antibleaching reagent.</a> Journal of the American Chemical Society. 131 (14), 5018-5019 (2009).</li><li>Aitken, C. E., Marshall, R. A., Puglisi, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+oxygen+scavenging+system+for+improvement+of+dye+stability+in+single-molecule+fluorescence+experiments.">An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments.</a> Biophysical Journal. 94 (5), 1826-1835 (2008).</li><li>Lee, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+do+short+chain+nonionic+detergents+destabilize+G-protein-coupled+receptors.">How do short chain nonionic detergents destabilize G-protein-coupled receptors.</a> Journal of the American Chemical Society. 138 (47), 15425-15433 (2016).</li><li>Cao, A. -. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allosteric+modulators+enhance+agonist+efficacy+by+increasing+the+residence+time+of+a+GPCR+in+the+active+state.">Allosteric modulators enhance agonist efficacy by increasing the residence time of a GPCR in the active state.</a> Nature Communications. 12 (1), 1-13 (2021).</li><li>Mancebo, A., Mehra, D., Banerjee, C., Kim, D. -. H., Puchner, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+cross-correlation+filtering+of+one-and+two-color+single+molecule+localization+microscopy+data.">Efficient cross-correlation filtering of one-and two-color single molecule localization microscopy data.</a> Frontiers in Bioinformatics. 1, 739769 (2021).</li><li>Mehra, D., Adhikari, S., Banerjee, C., Puchner, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+locus+specific+chromatin+structure+and+dynamics+with+correlative+conventional+and+super-resolution+imaging+in+living+cells.">Characterizing locus specific chromatin structure and dynamics with correlative conventional and super-resolution imaging in living cells.</a> Nucleic Acids Research. , (2022).</li><li>Chen, H., Puhl, H. L., Koushik, S. V., Vogel, S. S., Ikeda, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+FRET+efficiency+and+ratio+of+donor+to+acceptor+concentration+in+living+cells.">Measurement of FRET efficiency and ratio of donor to acceptor concentration in living cells.</a> Biophysical Journal. 91 (5), 39-41 (2006).</li><li>Gopich, I. V., Szabo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FRET+efficiency+distributions+of+multistate+single+molecules.">FRET efficiency distributions of multistate single molecules.</a> The Journal of Physical Chemistry B. 114 (46), 15221-15226 (2010).</li><li>Roy, R., Hohng, S., Ha, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+guide+to+single-molecule+FRET.">A practical guide to single-molecule FRET.</a> Nature Methods. 5 (6), 507-516 (2008).</li><li>Hellenkamp, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precision+and+accuracy+of+single-molecule+FRET+measurements-A+multi-laboratory+benchmark+study.">Precision and accuracy of single-molecule FRET measurements-A multi-laboratory benchmark study.</a> Nature Methods. 15 (9), 669-676 (2018).</li><li>Bronson, J. E., Fei, J., Hofman, J. M., Gonzalez, R. L., Wiggins, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+rates+and+states+from+biophysical+time+series:+A+Bayesian+approach+to+model+selection+and+single-molecule+FRET+data.">Learning rates and states from biophysical time series: A Bayesian approach to model selection and single-molecule FRET data.</a> Biophysical Journal. 97 (12), 3196-3205 (2009).</li><li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+structural+elements+of+the+T-box+riboswitch+drive+the+two-step+binding+of+the+tRNA+ligand.">Specific structural elements of the T-box riboswitch drive the two-step binding of the tRNA ligand.</a> Elife. 7, 39518 (2018).</li><li>Goodman, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistical+analysis+based+on+a+certain+multivariate+complex+Gaussian+distribution+(an+introduction).">Statistical analysis based on a certain multivariate complex Gaussian distribution (an introduction).</a> The Annals of Mathematical Statistics. 34 (1), 152-177 (1963).</li><li>Brown, R. B., Audet, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+techniques+for+single-cell+lysis.">Current techniques for single-cell lysis.</a> Journal of the Royal Society Interface. 5, 131-138 (2008).</li><li>Schamber, M. R., Vafabakhsh, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+sensitivity+modulation+in+the+calcium-sensing+receptor+via+electrostatic+tuning.">Mechanism of sensitivity modulation in the calcium-sensing receptor via electrostatic tuning.</a> Nature Communications. 13 (1), 2194 (2022).</li><li>Jain, A., Liu, R., Xiang, Y. K., Ha, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+pull-down+for+studying+protein+interactions.">Single-molecule pull-down for studying protein interactions.</a> Nature Protocols. 7 (3), 445-452 (2012).</li><li>Huang, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delineating+the+conformational+landscape+of+the+adenosine+A(2A)+receptor+during+G+protein+coupling.">Delineating the conformational landscape of the adenosine A(2A) receptor during G protein coupling.</a> Cell. 184 (7), 1884-1894 (2021).</li><li>Wingler, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiotensin+analogs+with+divergent+bias+stabilize+distinct+receptor+conformations.">Angiotensin analogs with divergent bias stabilize distinct receptor conformations.</a> Cell. 176 (3), 468-478 (2019).</li><li>Gordon, C. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactivity+of+biarylazacyclooctynones+in+copper-free+click+chemistry.">Reactivity of biarylazacyclooctynones in copper-free click chemistry.</a> Journal of the American Chemical Society. 134 (22), 9199-9208 (2012).</li><li>Kim, E., Koo, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomedical+applications+of+copper-free+click+chemistry:+In+vitro,+in+vivo,+and+ex+vivo.">Biomedical applications of copper-free click chemistry: In vitro, in vivo, and ex vivo.</a> Chemical Science. 10 (34), 7835-7851 (2019).</li><li>Pickens, C. J., Johnson, S. N., Pressnall, M. M., Leon, M. A., Berkland, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+considerations,+challenges,+and+limitations+of+bioconjugation+via+azide-alkyne+cycloaddition.">Practical considerations, challenges, and limitations of bioconjugation via azide-alkyne cycloaddition.</a> Bioconjugate Chemistry. 29 (3), 686-701 (2018).</li><li>Geng, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+mechanism+of+ligand+activation+in+human+calcium-sensing+receptor.">Structural mechanism of ligand activation in human calcium-sensing receptor.</a> Elife. 5, 13662 (2016).</li></ol>

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 time<sup class="xre...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 (<s...

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 ...

Here, we used single-molecule FRET, and site-specific protein labeling via a natural amino acid incorporation to study the conformational dynamics of mGluR2. This allows to study the protein dynamics without perturbing protein structure. Atomic structure provide a static image of a protein.However, single-molecule FRET enables us to visualize the full range of protein motion in real-time and solution. This method can be applied to study the dynamics of any protein. Moreover, a natural amino acid incorporation can be used to study protein-protein interactions, and the engineering of synthetic receptors.Sample loading and optimization of imaging requires patience and practice. To begin, mark holes to be drilled on the glass slide with a marker. Use a Dremel to drill small holes on the slide while the slide is dipped in water.Sonicate the slides and cover slips in acetone for 30 minutes in a bath sonicator at 23 degrees Celsius. Dispose of acetone from the slide and cover slip jars. Rinse thoroughly with water, and sonicate in five-molar potassium hydroxide for 30 minutes.Next, rinse the slides and cover slip with water, and sonicate in methanol for two minutes. Leave the jars filled with methanol until the amino salinization step. Pour freshly prepared amino salinization mixture into the slide and cover slip jars.Incubate, sonicate, and again incubate the slides and cover slips in an amino salinization mixture before discarding the solution. Next, wash the jars with methanol before filling them with water. Then rinse the slides with water.Dry them with air blow, and place them in the assembly boxes. Apply 60 milliliters of PEGylation solution to the slide, and place a cover slip on top of it. Mark the non-PEGylated side before storage.After incubation, gently disassemble, and rinse the slides and cover slips with water. Then dry them by blowing air, and place them in a sterile 50-milliliter tube with the PEGylated surface facing away from each other. After passaging HEK 293T cells with 0.05%trypsin-EDTA, seed the cells on poly-L/D-lysine cover slips.Place the standard media with AZP supplemented media for growing cells and metabotropic glutamate receptor 2, or mGluR2 expression. Then transfect the cells with a transfection reagent, and incubate for 24 hours before changing the medium with fresh AZP supplemented media. 20 minutes before labeling with alkyne cyanine dyes, wash the cover slips twice with warm Recording Buffer, or RB, and place them in a warm standard media with no AZP.Then remove the standard media and wash the cells with RB before adding the freshly prepared labeling solution. Incubate for 15 minutes at 37 degrees Celsius in the dark. After incubation, remove the labeling solution.Wash the cover slip containing transfected cells twice with the RB, and re-suspend in the same medium. Next, pellet the cells by spinning at 1, 000 g at four degrees Celsius for five minutes, and remove the supernatant before re-suspending the pellet in 80 to 130 microliters of the lysis solution. Break the pellet by pipetting.Then wrap it in foil, and place it on the rocker at four degrees Celsius for 30 minutes to one hour to lyse the cells. Pellet the insoluble fraction by centrifugation at 20, 000 g and four degree Celsius for 20 minutes. Then transfer the supernatant to a fresh cold tube, and store it on ice.Remove the slide and cover slip from the freezer, and allow them to warm at room temperature in the dark. Assemble the chamber by sandwiching the strips of double-sided tape between the slide and cover slip, ensuring the PEGylated surfaces form the interior of the flow chamber. Using a pipette tip, press the cover slip to ensure the tape is contacting the cover slip and slide.Apply epoxy to the edges of the slide. Apply 40 microliters of 500-nanomolar NeutrAvidin to each lane, and incubate inside a humidified dark box. After two minutes, wash each chamber lane with 100 microliters of T50 buffer.Then add 20 nanomolar biotinylated antibody solution, and incubate for 30 minutes before washing with 200 microliters of T50 buffer per lane. Turn on the computer and microscope. Then turn on the lasers to warm up.Next, turn on the EMCCD camera, and open the software. Mount the sample chamber on the microscope stage, and add the protein sample gradually to achieve approximately 400 molecules per field of view. Wash the chamber with 100 microliters of imaging buffer supplemented with 0.3 units of protocatechuate 3, 4-dioxygenase.Adjust the gain, acquisition rate, and laser powers to detect single-molecule fluorescence signals in both the donor and accepter channels. Then excite the donor, and acquire time traces until 80%of the donor molecules in the field of view are photobleached. At the end of the movie, turn on the 640-nanometer laser to directly excite the accepter until some of the molecules are photobleached.Change the field of view, and repeat the steps for excitation of donor and accepter to collect three movies per condition. For selecting single-molecule FRET traces of particle picking, select the traces where the total intensity of donor and accepter traces is stable over time. Then select the traces with anti-correlated changes in donor and accepter intensities.Select the donor and accepter molecules showing single-step photobleaching and traces more than five seconds long. Calculate the FRET deficiency. Finally, identify the conformational state by Hidden Markov Modeling, or HMM, by following the steps described in the manuscript.The labeling of AZP by cyanine dyes was achieved by a copper-catalyzed cycloaddition reaction, and resulted in effective plasma membrane labeling of 548UAA with the cell surface population labeled with donor and accepter fluorophores. In SDS-PAGE electrophoresis, a single band at 250 kilodalton was observed in HEK 293T cells expressing 548UAA, which coincided with the full-length dimeric mGluR2. Representative selective traces for multiple short-lived and long-lived for donor and accepter bleaching events are shown here.Conformational states were identified using HMM analysis. Transitions between discrete FRET states were extracted from the idealized fits, and plotted as a transition density heat map. The idealized FRET traces also yield information on dwell time for each identified confirmation.The single-molecule's traces with donor and accepter channel is shown here. Further, single molecule FRET reveals four conformational states of the mGluR2 cysteine-rich domain. Efficient peak preservation is essential to single-molecule pull-down, and care should be taken during this process.In addition, the oxygen-scavenging agent should be added immediately prior to imaging. This labeling method and single-molecule FRET experiments have been applied to multiple receptors, and have provided new insights into the mechanism of receptor activation and modulation of receptor function.

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Research

JoVE Journal

Biochemistry

3.0K visualizações.  Northwestern University. Aqui, usamos FRET de molécula única e marcação de proteína site-specific através de uma incorporação natural de aminoácidos para estudar a dinâmica conformacional do mGluR2. Isso permite estudar a dinâmica da proteína sem perturbar a estrutura da proteína. A estrutura atômica fornece uma imagem estática de uma proteína.No entanto, o FRET de molécula única nos permite visualizar toda a gama de movimentos de proteínas em tempo real e solução. Este método pode ser ap...