Fluorescence-based single-molecule techniques, mainly including fluorescence correlation spectroscopy (FCS) and single-molecule fluorescence resonance energy transfer (smFRET), are able to analyze the conformational dynamics and diversity of biological macromolecules. They have been applied to analysis of the dynamics of membrane proteins, such as membrane receptors and membrane transport proteins, due to their superior ability in resolving spatio-temporal heterogeneity and the demand of trace amounts of analytes. In this review, we first introduced the basic principle involved in FCS and smFRET. Then we summarized the labeling and immobilization strategies of membrane protein molecules, the confocal-based and TIRF-based instrumental configuration, and the data processing methods. The applications to membrane protein dynamics analysis are described in detail with the focus on how to select suitable fluorophores, labeling sites, experimental setup, and analysis methods. In the last part, the remaining challenges to be addressed and further development in this field are also briefly discussed.
SalonJ.A.LodowskiD.T.PalczewskiK.“The Significance of G Protein-Coupled Receptor Crystallography for Drug Discovery”. Pharmacol. Rev. 2011. 63(4): 901–937.
2.
PiscitelliC.L.KeanJ.De GraafC., et al.“A Molecular Pharmacologist's Guide to G Protein-Coupled Receptor Crystallography”. Mol. Pharmacol. 2015. 88(3): 536–551.
3.
CasiraghiM.PointE.PozzaA., et al.“NMR Analysis of GPCR Conformational Landscapes and Dynamics”. Mol. Cell Endocrinol. 2019. 484: 69–77.
4.
GrahlA.AbikoL.A.IsogaiS., et al.“A High-Resolution Description of β1-Adrenergic Receptor Functional Dynamics and Allosteric Coupling from Backbone NMR”. Nat. Commun. 2020. 11(1): 2216.
5.
SafdariH.A.PandeyS.ShuklaA.K., et al.“Illuminating GPCR Signaling by Cryo-EM”. Trends Cell Biol. 2018. 28(8): 591–594.
6.
CourniaZ.AllenT.W.AndricioaeiI., et al.“Membrane Protein Structure, Function, and Dynamics: A Perspective from Experiments and Theory”. J. Membr. Biol. 2015. 248(4): 611–640.
7.
HauserA.S.ChavaliS.MasuhoI., et al.“Pharmacogenomics of GPCR Drug Targets”. Cell. 2018. 172(1–2): 41–54.e19.
8.
RosenbaumD.M.RasmussenS.G.KobilkaB.K.“The Structure and Function of G-Protein-Coupled Receptors”. Nature. 2009. 459(7245): 356–363.
9.
HilgerD.MasureelM.KobilkaB.K.“Structure and Dynamics of GPCR Signaling Complexes”. Nat. Struct. Mol. Biol. 2018. 25(1): 4–12.
10.
GirardinF.“Membrane Transporter Proteins: A Challenge for CNS Drug Development”. Dialogues Clin. Neurosci. 2006. 8(3): 311–321.
11.
KonigJ.MullerF.FrommM.F.“Transporters and Drug–Drug Interactions: Important Determinants of Drug Disposition and Effects”. Pharmacol. Rev. 2013. 65(3): 944–966.
12.
FerreS.CasadoV.DeviL.A., et al.“G Protein-Coupled Receptor Oligomerization Revisited: Functional and Pharmacological Perspectives”. Pharmacol. Rev. 2014. 66(2): 413–434.
13.
WangY.BuggeK.KragelundB.B., et al.“Role of Protein Dynamics in Transmembrane Receptor Signaling”. Curr. Opin. Struct. Biol. 2018. 48: 74–82.
14.
KrichevskyO.BonnetG.“Fluorescence Correlation Spectroscopy: The Technique and Its Applications”. Rep Prog Phys. 2002. 65(2): 251–297.
TorresT.LevitusM.“Measuring Conformational Dynamics: A New FCS-FRET Approach”. J. Phys. Chem. B. 2007. 111(25): 7392–7400.
18.
FelekyanS.KalininS.SanabriaH., et al.“Filtered FCS: Species Auto- and Cross-Correlation Functions Highlight Binding and Dynamics in Biomolecules”. ChemPhysChem. 2012. 13(4): 1036–1053.
DijkmanP.M.CastellO.K.GoddardA.D., et al.“Dynamic Tuneable G Protein-Coupled Receptor Monomer-Dimer Populations”. Nat. Commun. 2018. 9(1): 1710.
21.
HellenkampB.SchmidS.DoroshenkoO., et al.“Precision and Accuracy of Single-Molecule FRET Measurements: A Multi-Laboratory Benchmark Study”. Nat. Methods. 2018. 15(9): 669–676.
22.
LeeN.K.KapanidisA.N.WangY., et al.“Accurate FRET Measurements Within Single Diffusing Biomolecules Using Alternating-Laser Excitation”. Biophys. J. 2005. 88(4): 2939–2953.
23.
WhiteS.H.WimleyW.C.“Membrane Protein Folding and Stability: Physical Principles”. Annu. Rev. Biophys. Biomol. Struct. 1999. 28: 319–365.
SchulzG.E.“Transmembrane Beta-Barrel Proteins”. Adv. Protein Chem. 2003. 63: 47–70.
26.
BlountP.IsclaI.“Life with Bacterial Mechanosensitive Channels, from Discovery to Physiology to Pharmacological Target”. Microbiol. Mol. Biol. Rev. 2020. 84(1): e00055–19.
27.
BoothI.R.BlountP.“The MSCS and MSCL Families of Mechanosensitive Channels Act as Microbial Emergency Release Valves”. J. Bacteriol. 2012. 194(18): 4802–4809.
28.
CherezovV.RosenbaumD.M.HansonM.A., et al.“High-Resolution Crystal Structure of an Engineered Human β2-Adrenergic G Protein-Coupled Receptor”. Science. 2007. 318(5854): 1258–1265.
29.
RasmussenS.G.DevreeB.T.ZouY., et al.“Crystal Structure of the β2 Adrenergic Receptor–Gs Protein Complex”. Nature. 2011. 477(7366): 549–555.
30.
LeeS.LeeJ.HohngS.“Single-Molecule Three-Color FRET with Both Negligible Spectral Overlap and Long Observation Time”. PLoS One. 2010. 5(8): e12270.
31.
GregorioG.G.MasureelM.HilgerD., et al.“Single-Molecule Analysis of Ligand Efficacy in β2 AR-G-Protein Activation”. Nature. 2017. 547(7661): 68–73.
32.
PatiA.K.El BakouriO.JockuschS., et al.“Tuning the Baird Aromatic Triplet-State Energy of Cyclooctatetraene to Maximize the Self-Healing Mechanism in Organic Fluorophores”. Proc. Natl. Acad. Sci. U.S.A. 2020. 117(39): 24305–24315.
33.
YangZ.T.LiL.J.LingJ., et al.“Cyclooctatetraene-Conjugated Cyanine Mitochondrial Probes Minimize Phototoxicity in Fluorescence and Nanoscopic Imaging”. Chem. Sci. 2020. 11(32): 8506–8516.
34.
JiaK.WanY.XiaA., et al.“Characterization of Photoinduced Isomerization and Intersystem Crossing of the Cyanine Dye Cy3”. J. Phys. Chem. A. 2007. 111(9): 1593–1597.
35.
LevitusM.RanjitS.“Cyanine Dyes in Biophysical Research: The Photophysics of Polymethine Fluorescent Dyes in Biomolecular Environments”. Q. Rev. Biophys. 2011. 44(1): 123–151.
36.
HwangH.KimH.MyongS.“Protein Induced Fluorescence Enhancement as a Single Molecule Assay with Short Distance Sensitivity”. Proc. Natl. Acad. Sci. U.S.A. 2011. 108(18): 7414–7418.
CooperM.EbnerA.BriggsM., et al.“Cy3B: Improving the Performance of Cyanine Dyes”. J. Fluoresc. 2004. 14(2): 145–150.
39.
SouliereM.F.AltmanR.B.SchwarzV., et al.“Tuning a Riboswitch Response Through Structural Extension of a Pseudoknot”. Proc. Natl. Acad. Sci. U.S.A. 2013. 110(35): E3256–E3264.
40.
SchulerB.PannellL.K.“Specific Labeling of Polypeptides at Amino-Terminal Cysteine Residues Using Cy5-Benzyl Thioester”. Bioconjugate Chem. 2002. 13(5): 1039–1043.
41.
WangL.FanJ.L.QiaoX.Q., et al.“Novel Asymmetric Cy5 Dyes: Synthesis, Photostabilities, and High Sensitivity in Protein Fluorescence Labeling”. J. Photoch. Photobio. A. 2010. 210(2–3): 168–172.
42.
KimY.HoS.O.GassmanN.R., et al.“Efficient Site-Specific Labeling of Proteins via Cysteines”. Bioconjug. Chem. 2008. 19(3): 786–791.
43.
TianH.FurstenbergA.HuberT.“Labeling and Single-Molecule Methods to Monitor G Protein-Coupled Receptor Dynamics”. Chem. Rev. 2017. 117(1): 186–245.
44.
NaranjoA.N.ChevalierA.CousinsG.D., et al.“Conserved Disulfide Bond is not Essential for the Adenosine A2A Receptor: Extracellular Cysteines Influence Receptor Distribution Within the Cell and Ligand-Binding Recognition”. Biochim. Biophys. Acta. 2015. 1848(2): 603–614.
45.
CookJ.V.EidneK.A.“An Intramolecular Disulfide Bond Between Conserved Extracellular Cysteines in the Gonadotropin-Releasing Hormone Receptor is Essential for Binding and Activation”. Endocrinology. 1997. 138(7): 2800–2806.
46.
ProbstW.C.SnyderL.A.SchusterD.I., et al.“Sequence Alignment of the G-Protein Coupled Receptor Superfamily”. DNA Cell Biol. 1992. 11(1): 1–20.
47.
NaumenkoV.S.PonimaskinE.“Palmitoylation as a Functional Regulator of Neurotransmitter Receptors”. Neural Plast. 2018. 2018: 5701348.
48.
ZhangX.LeH.T.ZhangX., et al.“Palmitoylation on the Carboxyl Terminus Tail is Required for the Selective Regulation of Dopamine D2 Versus D3 Receptors”. Biochim. Biophys. Acta. 2016. 1858(9): 2152–2162.
49.
WangY.LiuY.X.DebergH.A., et al.“Single Molecule FRET Reveals Pore Size and Opening Mechanism of a Mechano-Sensitive Ion Channel”. eLife. 2014. 3: e01834.
50.
AdamsS.R.CampbellR.E.GrossL.A., et al.“New Biarsenical Ligands and Tetracysteine Motifs for Protein Labeling In Vitro and In Vivo: Synthesis and Biological Applications”. J. Am. Chem. Soc. 2002. 124(21): 6063–6076.
51.
HoffmannC.GaiettaG.ZurnA., et al.“Fluorescent Labeling of Tetracysteine-Tagged Proteins in Intact Cells”. Nat. Protoc. 2010. 5(10): 1666–1677.
52.
MartinB.R.GiepmansB.N.AdamsS.R., et al.“Mammalian Cell-Based Optimization of the Biarsenical-Binding Tetracysteine Motif for Improved Fluorescence and Affinity”. Nat. Biotechnol. 2005. 23(10): 1308–1314.
53.
HoffmannC.GaiettaG.BunemannM., et al.“A FlAsH-Based FRET Approach to Determine G Protein-Coupled Receptor Activation in Living Cells”. Nat. Methods. 2005. 2(3): 171–176.
54.
JuilleratA.GronemeyerT.KepplerA., et al.“Directed Evolution of O6-Alkylguanine-DNA Alkyltransferase for Efficient Labeling of Fusion Proteins with Small Molecules In Vivo”. Chem. Biol. 2003. 10(4): 313–317.
55.
GautierA.JuilleratA.HeinisC., et al.“An Engineered Protein Tag for Multiprotein Labeling in Living Cells”. Chem. Biol. 2008. 15(2): 128–136.
56.
MaurelD.Comps-AgrarL.BrockC., et al.“Cell-Surface Protein–Protein Interaction Analysis with Time-Resolved FRET and Snap-Tag Technologies: Application to GPCR Oligomerization”. Nat. Methods. 2008. 5(6): 561–567.
57.
UttamapinantC.WhiteK.A.BaruahH., et al.“A Fluorophore Ligase for Site-Specific Protein Labeling Inside Living Cells”. Proc. Natl. Acad. Sci. U.S.A. 2010. 107(24): 10914–10919.
58.
LiuC.C.SchultzP.G.“Adding New Chemistries to the Genetic Code”. Annu. Rev. Biochem. 2010. 79: 413–444.
59.
HuberT.SakmarT.P.“Chemical Biology Methods for Investigating G Protein-Coupled Receptor Signaling”. Chem. Biol. 2014. 21(9): 1224–1237.
60.
RyuY.SchultzP.G.“Efficient Incorporation of Unnatural Amino Acids into Proteins in Escherichia coli”. Nat. Methods. 2006. 3(4): 263–265.
61.
WangK.SachdevaA.CoxD.J., et al.“Optimized Orthogonal Translation of Unnatural Amino Acids Enables Spontaneous Protein Double-Labelling and FRET”. Nat. Chem. 2014. 6(5): 393–403.
62.
ChatterjeeA.GuoJ.LeeH.S., et al.“A Genetically Encoded Fluorescent Probe in Mammalian Cells”. J. Am. Chem. Soc. 2013. 135(34): 12540–12543.
63.
LeeH.S.GuoJ.LemkeE.A., et al.“Genetic Incorporation of a Small, Environmentally Sensitive, Fluorescent Probe into Proteins in Saccharomyces cerevisiae”. J. Am. Chem. Soc. 2009. 131(36): 12921–12923.
64.
HilaireM.R.AhmedI.A.LinC.W., et al.“Blue Fluorescent Amino Acid for Biological Spectroscopy and Microscopy”. Proc. Natl. Acad. Sci. U.S.A. 2017. 114(23): 6005–6009.
65.
YeS.KohrerC.HuberT., et al.“Site-Specific Incorporation of Keto Amino Acids into Functional G Protein-Coupled Receptors Using Unnatural Amino Acid Mutagenesis”. J. Biol. Chem. 2008. 283(3): 1525–1533.
66.
HuberT.NaganathanS.TianH., et al.“Unnatural Amino Acid Mutagenesis of GPCRs Using Amber Codon Suppression and Bioorthogonal Labeling”. Methods Enzymol. 2013. 520: 281–305.
67.
NaganathanS.YeS.SakmarT.P., et al.“Site-Specific Epitope Tagging of G Protein-Coupled Receptors by Bioorthogonal Modification of a Genetically Encoded Unnatural Amino Acid”. Biochemistry. 2013. 52(6): 1028–1036.
68.
MeinekeB.HeimgartnerJ.EirichJ., et al.“Site-Specific Incorporation of Two NCAAs for Two-Color Bioorthogonal Labeling and Crosslinking of Proteins on Live Mammalian Cells”. Cell Rep. 2020. 31(12): 107811.
69.
NaganathanS.Ray-SahaS.ParkM., et al.“Multiplex Detection of Functional G Protein-Coupled Receptors Harboring Site-Specifically Modified Unnatural Amino Acids”. Biochemistry. 2015. 54(3): 776–786.
70.
SaalK.A.RichterF.RehlingP., et al.“Combined Use of Unnatural Amino Acids Enables Dual-Color Super-Resolution Imaging of Proteins via Click Chemistry”. ACS Nano. 2018. 12(12): 12247–12254.
71.
UrnerL.H.LikoI.YenH.Y., et al.“Modular Detergents Tailor the Purification and Structural Analysis of Membrane Proteins Including G-Protein Coupled Receptors”. Nat. Commun. 2020. 11(1): 564.
72.
LeeS.GhoshS.JanaS., et al.“How Do Branched Detergents Stabilize GPCRs in Micelles?”. Biochemistry. 2020. 59(23): 2125–2134.
73.
TianH.SakmarT.P.HuberT.“Micelle-Enhanced Bioorthogonal Labeling of Genetically Encoded Azido Groups on the Lipid-Embedded Surface of a GPCR”. ChemBioChem. 2015. 16(9): 1314–1322.
74.
MilicD.VeprintsevD.B.“Large-Scale Production and Protein Engineering of G Protein-Coupled Receptors for Structural Studies”. Front. Pharmacol. 2015. 6: 66.
75.
ChenI.HowarthM.LinW., et al.“Site-Specific Labeling of Cell Surface Proteins with Biophysical Probes Using Biotin Ligase”. Nat. Methods. 2005. 2(2): 99–104.
76.
KayB.K.ThaiS.VolginaV.V.“High-Throughput Biotinylation of Proteins”. Methods Mol. Biol. 2009. 498: 185–196.
77.
de BoerE.RodriguezP.BonteE., et al.“Efficient Biotinylation and Single-Step Purification of Tagged Transcription Factors in Mammalian Cells and Transgenic Mice”. Proc. Natl. Acad. Sci. U.S.A. 2003. 100(13): 7480–7485.
78.
FairheadM.HowarthM.“Site-Specific Biotinylation of Purified Proteins Using Bira”. Methods Mol. Biol. 2015. 1266: 171–184.
79.
EliaG.“Biotinylation Reagents for the Study of Cell Surface Proteins”. Proteomics. 2008. 8(19): 4012–4024.
80.
VafabakhshR.LevitzJ.IsacoffE.Y.“Conformational Dynamics of a Class C G-Protein-Coupled Receptor”. Nature. 2015. 524(7566): 497–501.
81.
OlofssonL.FelekyanS.DoumazaneE., et al.“Fine Tuning of Sub-Millisecond Conformational Dynamics Controls Metabotropic Glutamate Receptors Agonist Efficacy”. Nat. Commun. 2014. 5: 5206.
82.
D. Axelrod. “Total Internal Reflection Fluorescence Microscopy”. In: J.J. Correia, H.W. Dietrich, editors. Biophysical Tools for Biologists, Volume Two: In Vivo Techniques. Amsterdam: Elsevier Academic Press, 2008. Chap. 7, Pp. 169–221.
83.
KapanidisA.N.LeeN.K.LaurenceT.A., et al.“Fluorescence-Aided Molecule Sorting: Analysis of Structure and Interactions by Alternating-Laser Excitation of Single Molecules”. Proc. Natl. Acad. Sci. U.S.A. 2004. 101(24): 8936–8941.
84.
DooseS.HeilemannM.MichaletX., et al.“Periodic Acceptor Excitation Spectroscopy of Single Molecules”. Eur. Biophys. J. 2007. 36(6): 669–674.
85.
KapanidisA.N.LaurenceT.A.LeeN.K., et al.“Alternating-Laser Excitation of Single Molecules”. ACC Chem. Res. 2005. 38(7): 523–533.
86.
MargeatE.KapanidisA.N.TinnefeldP., et al.“Direct Observation of Abortive Initiation and Promoter Escape Within Single Immobilized Transcription Complexes”. Biophys. J. 2006. 90(4): 1419–1431.
87.
MullerB.K.ZaychikovE.BrauchleC., et al.“Pulsed Interleaved Excitation”. Biophys. J. 2005. 89(5): 3508–3522.
88.
LambD.C.MullerB.K.BruachleC.“Applications of Pulsed Interleaved Excitation”. Biophys. J. 2005. 88(1): 373a–373a.
89.
McKinneyS.A.JooC.HaT.“Analysis of Single-Molecule FRET Trajectories Using Hidden Markov Modeling”. Biophys. J. 2006. 91(5): 1941–1951.
90.
LamichhaneR.LiuJ.J.PljevaljcicG., et al.“Single-Molecule View of Basal Activity and Activation Mechanisms of the G Protein-Coupled Receptor β2AR”. Proc. Natl. Acad. Sci. U.S.A. 2015. 112(46): 14254–14259.
91.
HabrianC.H.LevitzJ.VyklickyV., et al.“Conformational Pathway Provides Unique Sensitivity to a Synaptic mGluR”. Nat. Commun. 2019. 10(1): 5572.
92.
AkyuzN.AltmanR.B.BlanchardS.C., et al.“Transport Dynamics in a Glutamate Transporter Homologue”. Nature. 2013. 502(7469): 114–118.
