Labeling and Single-Molecule Methods To Monitor G Protein


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Labeling and Single-Molecule Methods To Monitor G ProteinCoupled Receptor Dynamics He Tian,† Alexandre Fürstenberg,† and Thomas Huber* Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, 1230 York Avenue, New York, New York 10065, United States ABSTRACT: The superfamily of G protein-coupled receptors (GPCRs) mediates a wide range of physiological responses and serves as an important category of drug targets. Earlier biochemical and biophysical studies have shown that GPCRs exist temporally in an ensemble of interchanging conformations. Single-molecule techniques are ideally suited to understand the dynamic signaling and conformational complexity of G protein-coupled receptors (GPCRs). Here, we review the progress in single-molecule studies on GPCRs. We introduce the fundamental technical aspects of single-molecule fluorescence. We also survey the methodologies for labeling GPCRs with biophysical probes, particularly fluorescent dyes, and highlight the relevant chemical biology innovations that can be instrumental for studying GPCRs. Finally, we illustrate how the optical techniques and the labeling schemes have been combined to investigate GPCR signaling and dynamics at the single-molecule level.

CONTENTS 1. Introduction 2. The Era of Single Molecules in Biology 2.1. Why Single Molecules 2.2. Basic Requirements 2.3. Fluorescence Observables 2.4. Microscope Configurations: Spectroscopy or Imaging? 2.5. Observing Diffusing Molecules 2.6. Imaging Immobilized Molecules 2.7. Single-Molecule Trapping 2.8. Super-Resolution Imaging 2.9. Multicolor Single-Molecule Detection 3. Cell Biology and Biochemistry of GPCRs 3.1. GPCRs as Important Drug Targets 3.2. Assembly of the GPCR Signaling Complex 3.3. Spectroscopic and Structural Studies on GPCR Activation 3.4. Conformational Diversity of GPCRs 3.5. Membrane Dynamics and Oligomerization of GPCRs 4. Labeling of GPCRs with Biophysical Probes 4.1. Overview 4.2. How to Specifically Target a GPCR 4.3. Immunofluorescence Using Antibodies and Nanobodies 4.3.1. Antibodies 4.3.2. Nanobodies 4.4. Fluorescent Ligands 4.5. Ligand-Directed Labeling 4.6. Fluorescent Proteins and Fö rster Resonance Energy Transfer (FRET)

© XXXX American Chemical Society

4.7. Luciferase and Bioluminescence Resonance Energy Transfer (BRET) 4.8. Peptide-Based Tags 4.8.1. Arsenical Hairpin Binders Specific for the Tetracysteine Tag 4.8.2. Bisboronic Probe Specific for the Tetraserine Tag 4.8.3. Tetranuclear Zinc(II) Probe Specific for the Oligo-aspartate Tag 4.8.4. Template-Directed Labeling Based on a Coiled-Coil Motif 4.9. Chemoenzymatic Labeling Based on SelfLabeling Protein Tags 4.9.1. SNAP-Tag and CLIP-Tag 4.9.2. Halo-Tag 4.9.3. TMP-Tag 4.10. Chemoenzymatic Labeling Based on Posttranslational Modification Enzymes 4.10.1. Biotin Ligase and Lipoic Acid Ligase 4.10.2. Sortase 4.10.3. Formylglycine-Generating Enzyme 4.10.4. Ascorbate Peroxidase 4.10.5. Applications of the Engineered Posttranslational Enzymes 4.11. Classic Approach for Site-Specific Labeling of GPCRs 4.11.1. Targeting the Naturally Occurring Functionalities in GPCRs

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Chemical Reviews 4.11.2. Spectroscopic Studies on GPCRs Enabled by Cysteine and Lysine Labeling 4.11.3. Limitations to Targeting Naturally Occurring Functionalities 4.12. Novel Approaches for Site-Specific Labeling of GPCRs 4.12.1. Incorporating Unnatural Amino Acids into GPCRs 4.12.2. Genetically Encoded Unnatural Amino Acids as Biophysical Probes for GPCRs 4.12.3. Bioorthogonal Labeling of GPCRs Targeting Genetically Encoded Reactive Handles 4.12.4. Potential Issues with Amber Codon Suppression in Living Cells 4.13. Fluorogenic Labeling Reactions 4.14. Choosing the Right Labeling Method To Understand the Biochemistry and Cell Biology of GPCRs 4.14.1. Tracking GPCR Conformational Change 4.14.2. Trafficking and Internalization 4.14.3. Oligomerization 5. Application of Single-Molecule Methods to GPCRs 5.1. Mobility, Oligomerization, and Stoichiometry 5.2. Membrane Organization beyond the Diffraction Limit 5.3. Conformational Dynamics 5.4. Ligand Binding 5.5. Structure and Stability 6. Conclusion and Prospect Author Information Corresponding Author Author Contributions Notes Biographies Acknowledgments References

Review

facilitate the endeavor of drug discovery since a large proportion of existing therapeutic agents target GPCRs.1,2 Given the challenge of identifying effective drug targets, this classic approach to modulating GPCRs is unlikely to be ever out of fashion. To quote James Black, the British pharmacologist and Nobel laureate who was best known for developing βblockers targeting β-adrenergic receptors, “the most fruitful basis of the discovery of a new drug is to start with an old drug.” In the past decade, the GPCR field has witnessed an explosion of crystal structures, which has tremendously advanced the understanding of the structure−function relationship of the receptors.3,5 Crystallography is the reference method for providing high-resolution spatial information. Nonetheless, due to their static nature, crystal structures can only offer very limited insight into the dynamics of conformations and of signaling. Intrinsically, the crystallization of a protein eliminates its conformational diversity. Moreover, the extensive protein engineering required for crystallization often sacrifices the structural information on the flexible loops and termini. All of these considerations point to an acute need for developing alternative methodologies. Single-molecule techniques nicely complement crystallography in uncovering the dynamics and conformational complexity of the GPCR signaling complex. At the beginning of this Review, we describe the basic concepts in single-molecule fluorescence techniques. We also introduce several key events in GPCR signaling including the assembly of the GPCR signaling complex, receptor activation, receptor conformational diversity, and receptor oligomerization. A major technical challenge for single-molecule fluorescence experiments is to prepare suitable fluorescently labeled receptors. We therefore strive to provide an overview of the methodologies for attaching extrinsic probes to GPCRs, with an emphasis on fluorescent labeling. The enabling role of labeling strategies in spectroscopic and imaging experiments is illustrated, and the advantages and limitations of each strategy are discussed and compared. Many labeling schemes have been employed in ensemble measurements and may serve as the stepping-stones for single-molecule studies. We also examine recent innovations from chemical biology, such as liganddirected labeling, chemoenzymatic labeling, unnatural amino mutagenesis, bioorthogonal chemistries, fluorogenic reactions, and how these approaches can be transferred to the GPCR field. Relevant reviews are referenced to orient the GPCR specialists to explore the chemical biology toolkit. Finally, we review existing single-molecule studies on GPCRs and list them in an extensive table, with annotations specifying the techniques and summarizing the key findings.

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1. INTRODUCTION The G protein-coupled receptors (GPCRs) constitute a large family of transmembrane receptors that transduce extracellular signals into intracellular biochemical responses. GPCRs mediate a myriad of fundamental physiological processes, such as vision, smell, taste, neurotransmission, immune response, mood and behavioral regulation, homeostasis, metabolism, and energy balance. Three Nobel prizes have been awarded to scientists working on GPCRs: George Wald, who elucidated the biochemistry of the visual photoreceptor rhodopsin (physiology or medicine, 1967); Richard Axel and Linda Buck, who discovered olfactory receptors (physiology or medicine, 2004); and a few years ago Robert Lefkowitz and Brian Kobilka, who made groundbreaking contributions to understanding the molecular basis of GPCR function, in particular, that of adrenergic receptors (chemistry, 2012). Several other Nobel prizes were closely related to the GPCR signaling pathway. Understanding with molecular precision how GPCRs function in cells is an active area of research. In addition to a basic understanding of transmembrane signaling, studies of GPCRs can provide insights that might

2. THE ERA OF SINGLE MOLECULES IN BIOLOGY Single-molecule observation and manipulation in vitro and in living cells have been revolutionizing our way to address biological questions, enabling scientists to monitor elementary biochemical reactions with an unmatched level of detail and to capture images of ongoing processes in living cells at an unprecedented resolution. More than 25 years ago, the first optical detection experiments on single molecules, first at 4 K,6 then at room temperature,7 were mostly driven by the curiosity of physicists. Since then, single-molecule spectroscopy and imaging have emerged as routine techniques in biological research.8 Many areas have benefited from the information-rich high-quality data, which can nowadays be collected both on B

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readers of some basic requirements (Figure 2). Single-molecule fluorescence measurements traditionally require a transparent,

purified components in vitro or by following individual molecules in living cells.9 2.1. Why Single Molecules

Populations of biomolecules are most of the time heterogeneous. For example, the individual copies of the same protein or nucleic acid strand are in different conformations or at different stages of the enzymatic cycle. The main motivation for using single-molecule methods is to measure the full distributions of behavior and to expose hidden heterogeneities that would be totally obfuscated if only the population average were evaluated (which is the case in any measurement of an ensemble of molecules). The shape of the distribution might be skewed or reveal distinct subpopulations of molecules (Figure 1), potentially offering mechanistic insight. Furthermore, singleFigure 2. Excitation of a single molecule. (a) Typical Jablonski diagram for single-molecule fluorescence spectroscopy and imaging. A typical organic fluorescent probe is excited into vibrationally excited levels of its first excited state. After fast vibrational relaxation (VR) to the ground vibrational level of the excited electronic state, the molecule may either emit fluorescence, relax nonradiatively to its ground state via internal conversion (IC), or undergo intersystem crossing (ISC) to a nonemissive triplet state. (b) Schematic illustration of a focused optical beam exciting a single molecule on a coverslip surface. Key parameters for a good single-molecule fluorescent probe are molar extinction coefficient (ε), fluorescence quantum yield (Φfl), photostability, characterized by its photobleaching quantum yield (Φb), blinking characteristics, and linkability, that is, the ability to be selectively attached to molecules of interest.

Figure 1. Populations in single-molecule spectroscopy. Fluorescent lipids freely diffuse in a bilayer (left) but sometimes stop at a particular spot before starting diffusing again. An analysis of their motion reveals two populations: one with a high diffusion coefficient corresponding to the moving molecules, and another with a low diffusion coefficient corresponding to the immobilized molecules. A simple measurement of the average diffusion coefficient would, however, yield a value falling somewhere between the two populations, failing to represent the true molecular behaviors. Scale bar in the left panel: 5 μm.

nonfluorescent host matrix (crystal, polymer, solvent, cell) deposited on a thin coverslip made out of low fluorescence glass. In such a matrix, the molecules are observed at concentrations low enough for individual molecules to be separated in space (more than the diffraction limit of ∼200 nm), time, or wavelength.23 Apart from a few exceptions, biological molecules are optically undetectable unless coupled to a probe. A useful probe, most of the time a fluorophore, should have the following general properties: (1) absorbing light efficiently (high extinction coefficient); (2) emitting light efficiently (high fluorescence quantum yield); (3) displaying high photostability (the total number of photons emitted before photobleaching is large); and (4) specifically linkable to the molecule of interest. A steady flow of emitted photons with rare “blinking” events (fluorescence intermittence) is also highly desirable for most applications, except for singlemolecule based super-resolution imaging. Selective excitation is usually achieved using a laser beam resonant with the optical transition of the probe. Cellular autofluorescence limits singlemolecule detection to excitation with wavelengths typically larger than about 480 nm.

molecule measurements can follow the internal states of the same molecule over time and the transitions between them, thus revealing rare intermediate states or hidden kinetic pathways and circumventing the need for sample synchronization. Stochastic multistep processes, such as the dance of individual molecular motors or tRNA transit on single translating ribosomes, could be monitored.10,11 Typical singlemolecule labels behave like nanometer-sized light sources that are strongly influenced by their close surroundings. They are therefore local reporters of the local environment and provide a direct window into the nanoscale and its changes over time. A surprising observation from the early single-molecule experiments was precisely the changes in the fluorescence excitation or emission spectrum of a single molecule over time, a phenomenon termed “spectral diffusion” indicative of variations in the proximal environment of the probe.12−14 Remarkably, optical excitation enables observation of exactly one molecule surrounded by innumerable other transparent molecules composing a crystal, a polymer matrix, the solvent, or a cell. Therefore, single-molecule spectroscopy reaches the ultimate limit of sensitivity of ∼1.66 × 10−24 moles of the molecule of interest (1.66 yoctomoles). Being able to discriminate the signal arising from one, two, or a few molecules, single-molecule spectroscopy and imaging have found quantitative applications, including molecular counting or the determination of molecular stoichiometries.

2.3. Fluorescence Observables

In single-molecule fluorescence experiments, the primary role of the fluorophore is, of course, to reveal the existence of the molecule of interest as well as its position (Figure 3a). Multiple fluorophores of different colors can be simultaneously detected by sorting the emitted photons into different channels on the basis of their wavelengths. This multicolor detection scheme can be used to monitor colocalization of different molecules (Figure 3b). The location of a single fluorophore can be determined with nanometer precision in the laboratory frame (Figure 3c), enabling its trajectories to be reconstructed and the diffusional property to be evaluated.

2.2. Basic Requirements

The fundamental principles of single-molecule optical spectroscopy and imaging have been described extensively in many excellent reviews.8,9,15−22 Here, we will briefly remind the C

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Figure 3. Different processes that can be monitored by fluorescence spectroscopy and imaging at the single-molecule level. (a) A fluorophore (green) attached to a macromolecule reports its location, which can be determined within a few tens of nanometers. (b) Two molecules labeled with two different nonoverlapping fluorophores can be colocalized (green and red). (c) Localizing a single molecule over time can be used to track its moving trajectory and evaluate its diffusion properties. (d) The fluorescence intensity (I) of a single molecule going in and out of the excitation/ detection volume fluctuates over time (t) between a defined fluorescence level (arbitrary unit 1) and the background level. (e) Single-step photobleaching of noninteracting fluorophores enables the stoichiometry to be determined by counting the steps. (f) The orientational motion of a tethered fluorophore can be determined by using polarized excitation and detecting the polarization of the emission in two orthogonal channels (s and p). (g) Changes in the local environment of a fluorophore lead to fluctuations in the fluorescence intensity as well as lifetime. (h) Photoinduced electron transfer (PET) between a fluorophore and a quencher (Q) also modulates the fluorescence signal. (i) FRET between a donor (D) and an acceptor (A) fluorophore leads to anticorrelated changes in the donor and acceptor emission intensities.

Observing the intensity of a fluorescent spot over time can be informative by itself. For example, the binding dynamics of a fluorescent molecule to its immobilized partner can be monitored (Figure 3d). Similarly, the stoichiometry and the copy number of molecules within a complex can be determined by following stepwise photobleaching events and comparing the total intensity of a complex with the intensity of a single fluorophore (Figure 3e).24,25 In addition to the fluorescence intensity, other fluorescence parameters can provide valuable information on the dynamics of biomolecules.26 First, the polarization of the emission of single fluorophores reflects their rotational freedom (Figure 3f) that can be altered by intermolecular interaction or changes in its local environment (e.g., viscosity). Second, the fluorescence lifetime (typically a few nanoseconds) of a single molecule also changes with the local environment of the dye. It is worth noting that a change in fluorescence lifetime is commonly, but not necessarily, correlated with changes in the fluorescence intensity.14,27 Because more than one mechanism may change in the fluorescence intensity and lifetime, the interpretation is

not always straightforward (Figure 3g). An important mechanism is photoinduced electron transfer (PET), through which the lifetime of a fluorescent molecule can be shortened by a proximal quencher moiety (Figure 3h).28−30 Some amino acid residues, such as tyrosine or tryptophan, can serve as such PET quenchers. Therefore, fluorescence lifetime is useful in probing protein intramolecular conformational changes or intermolecular interaction.31 Finally, if two different fluorophores come close enough, dipole−dipole interaction between them results in Förster resonance energy transfer (FRET) (Figure 3i). Because the efficiency of the energy transfer process is sensitive to the distance between the donor and acceptor, single-molecule FRET (smFRET) is a powerful tool for detecting the conformational changes or molecular interactions involving macromolecules. We will further discuss smFRET in section 2.9. 2.4. Microscope Configurations: Spectroscopy or Imaging?

Single-molecule experiments are generally performed on inverted fluorescence microscopes configured either in confocal D

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Figure 4. Typical microscope configurations in single-molecule fluorescence detection experiments.

Figure 5. Fluorescence correlation spectroscopy (FCS) and single-particle tracking (SPT). (a) FCS of single diffusing molecules. Diffusion of fluorescent molecules through an excitation laser beam leads to fluctuations in the fluorescence intensity over time. An autocorrelation analysis of the fluorescence intensity time trace reports on the time scales of the fluctuations and enables one to extract the diffusion coefficient τD of the studied species. (b) SPT. Movies of diffusing single molecules are recorded and the position of each molecule is subsequently determined with subpixel resolution in every frame. Determining positions of single molecules throughout the frames enables one to analyze individual trajectories and to extract the mean squared displacement (MSD) of the individual molecules. The shape of the MSD curve depends on the diffusing behavior of the molecule of interest and can, for example, distinguish cases where a molecule undergoes pure random Brownian motion (red) or is confined to a given area (green).

charge coupled device (EMCCD) or a scientific complementary metal−oxide−semiconductor (sCMOS) camera, to directly generate an image without the need to scan. Many fluorophores can be observed with low excitation intensity (typically on the order of a few W/cm2) at video rate, enabling translation of single molecules to be observed in real time. In a standard epifluorescence configuration, a large volume of the sample is excited so that background signal from out-of-focus emitters may severely affect imaging quality, especially for thicker samples. This problem is solved by the total-internal-reflection fluorescence (TIRF) configuration, in which a region less than 0.1 μm in thickness (as compared to 100 μm, or more, in epifluorescence) is selectively excited by the evanescent field of a totally internally reflected laser beam entering the objective off-center. TIRF is, however, only useful when the molecules of interest are immobilized on the surface of the coverslip, or confined to a narrow space above it. The confocal configuration achieves a greatly enhanced signal-to-noise ratio relative to wide-field imaging. The diffraction-limited illumination volume results in high excitation intensity (typically a few kW/cm2 on average), while the introduction of a pinhole greatly reduces the background. Furthermore, the temporal resolution of PMT or APD detectors is orders of magnitude better than that of cameras used in wide-field illumination, whose temporal resolution is limited to milliseconds by the necessity to read out a twodimensional detector array. Therefore, confocal point detection is preferred in spectroscopy experiments on single molecules. However, when it comes to observing dynamic structures, imaging large regions of interest, or tracking single molecules over time, wide-field configurations are clearly advantageous

scanning or in wide-field illumination mode (Figure 4). An infinity-corrected, high-numerical-aperture objective lens is used to excite the sample as well as to collect fluorescence emission. High-quality optical beam splitters and filters are required to separate the excitation and emission light, because the emitted photon flux from single fluorescing molecules is many orders of magnitude weaker than that of the excitation light.17 In a confocal configuration, the sample is illuminated with the smallest possible spot, which is achieved by focusing a collimated laser beam with the microscope objective to produce a diffraction-limited spot at the sample plane. The size of the illumination spot depends on the wavelength (λ) of the light and on the numerical aperture (NA) of the objective (a description of how much light the objective is designed to collect; typical oil-immersion objectives have NA values between 1.4 and 1.5). The dimension of the illumination spot can be estimated using Abbe’s limit for spatial resolution (∼0.5λ/NA, approximately 200 nm for visible light). The collected filtered emission is focused through a pinhole, which rejects out-of-focus light and thereby minimizes background photons that reach a point detector, like an avalanche photodiode (APD) or a photomultiplier tube (PMT). Moving the excitation light or the sample can cover a larger region of interest. Another class of single-molecule techniques uses wide-field microscopy. Here, the excitation source (usually a laser) is focused onto the back aperture of the objective to illuminate an area typically tens of micrometers in diameter. Fluorescence photons across the illuminated region are collected with a twodimensional array detector, such as an electron-multiplying E

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recognizes epitope-tagged molecules of interest, for example, GPCRs (Figure 6).39−41

because they allow different regions of the sample to be imaged simultaneously in real time. 2.5. Observing Diffusing Molecules

Molecules are not static in solution; they undergo constant Brownian motion. When a fluorescent molecule diffuses through a focused (confocal) laser beam, it gives a short fluorescence burst that lasts only up to a few milliseconds. By calculating the autocorrelation of the fluorescence intensity fluctuations over time arising from diffusing molecules, fluorescence correlation spectroscopy (FCS, Figure 5a) extracts information about average diffusion coefficients and molecular rotation predominantly at the origin of these fluctuations. Because the intensity of the autocorrelation function depends on the number of molecules present in average in the confocal volume, typically about one femtoliter, concentrations of samples in the picomolar to the nanomolar range can be determined by FCS.32 Although at a sufficiently low concentration the fluorescence fluctuations recorded in FCS experiments truly arise from single molecules, the outcome of such measurements is still the average value describing the entire ensemble. To characterize the underlying distribution of diffusional behavior, single fluorescent molecules are followed in real-time by wide-field fluorescence microscopy using the single-particle tracking (SPT) technique (Figure 5b).33 The fluorescent molecules are localized in a series of recorded frames. The two- or threedimensional trajectories can be reconstructed by comparing the adjacent frames. Diffusion coefficients are extracted by evaluating the mean squared displacement (MSD) of the particle over time. Such diffusion analysis can be used to classify the type of motion that the fluorescent molecule has undergone: purely Brownian (freely diffusing), directed (as is the case when there is flow or if a protein travels along the cytoskeleton of a cell), confined to an area, or immobile. The comparison of many individual trajectories of one type of molecules has given insights into the spatial or temporal heterogeneities of biological processes. For example, the diffusion coefficient of glycine receptors was found to depend on their location in the neuronal somatodendritic membrane and to change over time.34

Figure 6. Example of a general surface passivation and capturing scheme. The coverslip surface is coated with a silane layer to which biotinylated BSA is then cross-linked. The biotin (B) can bind an avidin protein (A), which in turn recognizes a biotinylated antibody. The latter recognizes a specific epitope of the protein of interest, in the case of this cartoon a GPCR embedded in a detergent micelle and sitespecifically labeled with a single fluorophore.

A second widely used passivation method involves silanization of the glass surface. Typical bifunctional silanization reagents bind covalently to the glass through an organofunctional alkoxysilane group. A large variety of organofunctional groups are available for modifying the surface property of glass. For example, aliphatic hydrocarbon or polyethylene glycol/ polyoxyethylene (PEG/POE) chains render the surface hydrophobic or hydrophilic, respectively.42,43 POE detergents, such as Tween-20, are used together with hydrophobic silanes to prepare hydrophilic glass surfaces,44 which efficiently prevent sticking of some biomolecules. These hydrocarbon or PEG/ POE chains can be further functionalized with chemically reactive groups to attach biomolecules of interest by amine- or thiol-selective reactions.

2.6. Imaging Immobilized Molecules

The time scale of biological reactions varies from milliseconds to seconds, and it is often desirable to observe a stationary molecule as long as possible. Several strategies have been developed to immobilize molecules to the surface of a coverslip.35 One way is to embed them into a polymer matrix such as gelatin, poly(vinyl alcohol) (PVA), or poly(methyl methacrylate) (PMMA).36,37 However, many biomolecules exhibit natural affinities for glass and can be captured on a surface simply by immersing a precleaned coverslip in a dilute solution. The problem is that impurities may also stick to the coverslip surface. Different methods have been devised to prevent nonspecific binding. Typically, a bifunctional passivation layer is required, which on one hand blocks the glass surface and prevents undesirable binding of nontarget molecules, and on the other hand specifically captures the target molecules. A popular choice of passivation layer is biotinylated bovine serum albumin (BSA) that nonspecifically adsorbs to the glass.38 Biotin binds to tetravalent avidins with high affinity and very slow dissociation rate. Avidins serve as the intermediate layer to connect the biotinylated BSA and the biotinylated target, or a biotinylated antibody that specifically

2.7. Single-Molecule Trapping

Surface immobilization by chemical means enables prolonged observation of biomolecules, but doubts persist regarding whether surface-attached molecules behave the same as molecules freely diffusing in solution. When the aim is to monitor subtle conformational changes and dynamics, additional control experiments are often required. The antiBrownian electrokinetic (ABEL) trap developed by Cohen and Moerner has made it possible to hold individual biomolecules in solution for up to several seconds without altering their internal degrees of freedom (Figure 7).45−47 It combines fluorescence-based position estimation with fast electrokinetic feedback to counter Brownian motion of a single F

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reduces the size of the microscope point spread function (PSF) by using patterned excitation beams and nonlinear response effects.56,57 The second class of imaging methods based on localization of single photoswitchable fluorescent molecules has become particularly popular due to their relative ease of implementation and the nature of qualitative and quantitative answers they can offer.58−61 These methods may be regrouped under the term of single-molecule localization microscopy (SMLM, Figure 8).62

Figure 7. Principle of the ABEL trap. In conventional FCS experiments, molecules freely diffuse in solution and give rise to very short fluorescence bursts as they pass through the excitation beam (trap off). When the electrokinetic feedback of the ABEL trap is turned on, individual fluorescent molecules are maintained within the excitation volume in a microfluidic cell (left) due to fast position readout and application of voltage pulses via four electrodes. Figure adapted and reproduced from ref 48. Copyright 2012 American Chemical Society.

molecule. The molecule is thereby maintaining its position in the field of view. ABEL trapping can be viewed as a form of real-time electrophoresis. Such a trap also applies to noncharged molecules of interest by taking advantage of the electroosmotic effect, that is, the ability of the trap to create a flow by dragging ions in solution.48 Because the molecule is kept in a region of time-averaged uniform intensity, the fluctuations in the fluorescence intensity can be informative. Conformational dynamics of DNA and proteins on the millisecond to second time scale, as well as molecular stoichiometries, have been successfully monitored with an ABEL trap.27,49,50 Interestingly, the photodynamics of allophycocyanin protein molecules trapped in solution differed substantially from the cases where the protein was either attached to a surface or embedded in polymers.51

Figure 8. Principle of single-molecule localization microscopy. (a) Fluorophores must be photoswitched between a dark and an emitting state. (b) Single molecules can be localized in individual frames and their position determined with nanometer accuracy by approximating their intensity profile by a mathematical function (usually a twodimensional Gaussian). The scale bar represents 1 μm. (c) By repeating the process over many thousands of frames, a superresolution image can be reconstructed from the list of molecular coordinates in a pointillist fashion.

The principle underlying SMLM is the realization that the center of the PSF originating from a single molecule (the “peak” of the fluorescence “mountain”) can be localized with much better accuracy than the width of the PSF itself.63,64 The localization precision (σ) of a single emitter is statistically related to the number of photons (N) detected from that emitter (σ ∝ N−1/2). Typically, the localization precision is on the order of 10−50 nm. SMLM relies on three basic principles to obtain a super-resolved image (Figure 8): first, fluorescent probes that can be photoswitched actively or stochastically between a fluorescent and a dark state; second, the repeated detection of many isolated emitters; and third, the reconstruction of a super-resolution image from the singlemolecule localization data. In each imaging frame, only a low number of molecules is excited to the fluorescent state so that single emitters can be spatially resolved and localized despite a high density of labeling. These simple principles can be implemented in various ways,62,65,66 and have been used to obtain static and dynamic structures of many biological and nonbiological samples.67,68 SMLM can also be used to determine the relative or absolute molecular copy numbers and cluster sizes.69,70 Furthermore, the direct output of SMLM is not an image but a list of molecular localizations and intensities. This form of data intensities has enabled direct coordinate-based data analysis schemes that would be impossible to realize with the pixelated intensity information generated by conventional optical microscopy.69,70

2.8. Super-Resolution Imaging

The size of a typical fluorescent label may vary from ∼1 nm for a single organic fluorophore, to 2−3 nm for a fluorescent protein, and up to 6−8 nm for the core of a luminescent nanocrystal (“quantum dot”). No matter how good the optical microscope is, all of these point-like objects appear at least ∼200 nm in size. This spot size in an image is determined by the diffraction of light that fundamentally limits the resolution (d) to about one-half the wavelength (λ) of the light (see eq 1), even with high-numerical-aperture (NA = n sin θ) optics. Initial attempts to achieve subdiffraction resolution involved near-field imaging, which used an aperture much smaller than the wavelength of light and thus only detects the emission light leaking through this tiny hole. Near-field scanning optical microscopy (NSOM) yielded the first success in imaging a single fluorescent molecule at room temperature.52 Nonetheless, the widespread application of NSOM has been hampered by its fundamental limitations related to the low-intensity light throughput through the very small aperture, the difficulty of implementation, and the actual short-range of the near-field that prevented it from becoming a widespread subdiffraction imaging technique.53 d = λ /(2n sin θ )

(1)

Two other approaches for overcoming the diffraction limit of light were theorized in the mid-1990s54,55 and experimentally demonstrated about a decade later, resulting in the recent boom of so-called super-resolution methods. The first class, exemplified by stimulated emission depletion (STED) microscopy and structured illumination microscopy (SIM), directly

2.9. Multicolor Single-Molecule Detection

An early development crucial for addressing increasingly complex mechanistic questions was the introduction of multiple G

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excitation wavelengths and multiple detection channels. Multichannel detection can nowadays easily be achieved using appropriate lasers and filter sets. Multicolor imaging has been applied to all methods described above. In particular, highresolution colocalization of single molecules can be achieved by imaging two different types of fluorophores separately and localizing them to nanometer accuracy.71,72 Both intermolecular and intramolecular interactions can thereby be monitored on the single-molecule level with a resolution an order of magnitude better than what can be achieved by confocal microscopy. Multicolor detection makes a significant transition from simple colocalization of the different fluorescent spots to FRET once the two different fluorophores come close enough ( 100 M−1 s−1)511 as compared to the sites on water-exposed surfaces, or the literature value (k2 = 0.1−1 M−1 s−1).521,536 This observed rate enhancement was attributed to the amphiphilic nature of the labeling reagent, which is comprised of a hydrophobic cyclooctyne and a hydrophilic dye, resulting in partitioning of the labeling reagent into the micelles. Specifically, the cyclooctyne partitions into the hydrocarbon core of the detergent micelle where it results in a high local reactant concentration. This micelle-enhanced SpAAC reaction, first observed from labeling GPCRs, was supported by a later report involving small molecules.541 The experiences with labeling the CCR5 receptor provided similar insights. The exposed extracellular and intracellular regions were better labeled with the Staudinger reaction.470 In contrast, a residue located deep in a hydrophobic binding pocket was readily modified by SpAAC.471 Taken together, these findings shed light on how the local environment on the protein surface can modulate the efficiencies of protein labeling. The capability of targeting the TM region of GPCRs complements the cysteine labeling chemistry that mostly targets the intracellular region. 4.12.3.2.5. Applications of Site-Specific Bioorthogonal Labeling of GPCRs. Bioorthogonal fluorescent labeling of azF-tagged GPCRs enables new FRET-based assays to investigate the GPCR signaling complex. Alexa Fluor 488labeled rhodopsin was exploited in a fluorescence-quenching assay for monitoring the formation of mature pigments.510,511 The ghrelin receptor labeled with Alexa Fluor 647 at the extracellular end of TM4 facilitated the study of ligand binding (Figure 12k).512 The ghrelin receptor tagged with Alexa Fluor 488 at the intracellular end of TM1 was used to study the assembly of receptor−G protein complex (Figure 12m).474 In AB

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Figure 23. Bioorthogonal labeling reactions with pyrrolysine analogues. (a) “Photoclick” chemistry between genetically encoded acrylamide (12, AcrK) and tetrazole.451 (b) “Photoclick” chemistry between genetically encoded cyclopropene (13, CpK) and tetrazole.545 (c) Cyanobenzothiazole condensation with genetically encoded 1,2-aminothiol (14, ThiPK).546 (d) Strain-promoted azide−alkyne [3+2] cycloaddition (SpAAC) between genetically encoded cyclooctynes (15, CoK1; 16, CoK2; 17, BCNK).547,548 (e) Strain-promoted inverse-electron-demand Diels−Alder cycloaddition (SPIEDAC) between genetically encoded cyclooctyne and tetrazine.553 (f) SPIEDAC between strained alkenes (18, NorK1; 19, NorK2; 20, TCOK) and tetrazine.550−553

4.12.3.4. Bioorthogonal Labeling of uaa-Tagged GPCRs on the Cell Surface. Fluorescent labeling of uaa-tagged GPCRs on the surface of live cells has not been demonstrated, but can be readily envisioned. The difficulty of achieving good contrast for on-cell labeling depends on several factors: first, the expression level of the uaa-tagged GPCRs; second, the nonspecific reactivity of the labeling reagent that results in a covalent bond with the native chemical functionalities in cells; third, the nonspecific, noncovalent binding between the labeling reagent and the cell surface. GPCRs are relatively low expressing on the mammalian cell surface (103−106 copies/cell). It should be kept in mind that bioorthogonal chemistries have high, but not absolute, selectivity over the native functionalities. In fact, strained alkenes554 and strained alkynes539 were shown to react with thiols. The cross-reactivity between tetrazines and thiols has also been suggested.555 On the cell surface the abundance of cysteines (>108 copies/cell) is orders of magnitude higher than that of even a high-expressing GPCR.510 For example, in the case of SpAAC between azF and dibenzocyclooctyne, its selectivity factor for azF over cysteine is between 200:1 and 800:1. Even if only 1/10 of the cysteines in membrane proteins were available for modification by cyclooctyne, the resulting signal-to-noise ratio would be far from ideal. The hydrophobic binding between cyclooctyne and the lipid bilayer of the plasma

all of these examples, the freedom of choosing the labeling site allowed rational design of the energy transfer scheme. 4.12.3.3. Targeting Genetically Encoded Pyrrolysine Analogues. The rapid progress in the pyrrolysyl-tRNA/aaRS system has enriched the toolkit of genetically encodable bioorthogonal reactive handles (Figure 22, Figure 23). Successful examples include (but not limited to): aliphatic alkynes (AlkK, 28),542 aliphatic terminal alkynes (azK, 29),542,543 acrylamide (AcrK, 30),451,544 cyclopropene (CpK, 31),545 1,2-aminothiols (ThiPK, 32),546 strained alkynes (CoK1, 33; CoK2, 34; BCNK, 35),547−549 strained alkenes (NorK1, 36; NorK2, 37; TCOK, 38),550−552 etc. Some of these newly developed labeling methods exhibit ultrafast reaction kinetics, particularly the strain-promoted inverse-electrondemand Diels−Alder cycloaddition (SPIEDAC) between tetrazine and BCNK (Figure 23e; k2 = 103−104 M−1 s−1), or between tetrazine and TCOK (Figure 23f; k2 > 104 M−1 s−1).513,552,553 While these fast labeling reactions await experimental demonstration for GPCRs, they should be particularly useful for labeling receptors with a short lifetime. A potential issue is that some genetically encoded reactive handles like strained alkynes or alkenes may suffer from crossreactivity with proximal cysteines. Hence, the labeling site may require optimization. AC

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Figure 24. Fluorogenic labeling strategies. (a) Fluorogenic reaction scheme 1: the dye bears a quenching reactive group and becomes highly fluorescent when the reaction alters the quenching group. (b) Azide as the quencher. The fluorescence signal is turned on by reaction with terminal or strained alkynes. (c) Tetrazine as the quencher. The dye is turned on by reaction with strained alkynes or alkenes. (d) Examples of fluorogenic dyes: hydroxycoumarin-azide,561 HELIOS-400Me tetrazine,667 Oregon Green-tetrazine,564 BODIPY-tetrazine,568 fluorescein derivative-azide,566 Sirhodamine derivative-azide.567 (e) Fluorogenic reaction scheme 2: the reaction causes the quencher to dissociate. (f) SNAP-tag for fluorogenic labeling. The fluorophore is attached to the benzyl group and the quencher to guanine. In BG-DY549-QSY7, QSY7 quenches the fluorescence of DY549 by 98%,569 corresponding to 50-fold signal enhancement. (g) TMP-tag for fluorogenic labeling. The first TMP-based fluorogenic label, TMPQ-Atto520, exhibits 20-fold signal enhancement upon covalent binding.311 AD

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reinitiation may result in a correctly folded product that only misses a fragment in the N-terminal domain but still covers the rest of the original sequence.469 These two scenarios may give rise to “ghost” receptors that escape fluorescent labeling but still signal. Therefore, it is essential to validate, with the most sensitive assay available, that the amount of untagged receptor is truly irrelevant to the biological question at hand.

membrane only exacerbates the problem. This estimation suggests that using cyclooctyne reagents to label and image low-abundance molecules on living cells is challenging. Another issue to bear in mind is that GPCRs, like other membrane proteins, do not stay indefinitely at the cell membrane. Their surface residence time ranges from minutes to hours.175 While endocytosis blockers or reduced temperature can be used to prolong the residence time, they may interfere with subsequent experiments. The fast labeling chemistry targeting genetically encoded strained alkenes or alkynes may represent a solution for on-cell labeling. The azide and tetrazine moieties are less hydrophobic than cycloocytnes, which helps to reduce nonspecific binding to the membrane. Furthermore, azide and tetrazine fluorescent labeling reagents can be fluorogenic, in other words, exhibiting dramatic signal enhancement upon conjugation. The strategies for developing fluorogenic labeling reagents will be discussed in greater details in section 4.13. Fluorogenic labeling of genetically encoded strained alkenes or alkynes has been demonstrated for heterologously expressed insulin receptor.556 4.12.4. Potential Issues with Amber Codon Suppression in Living Cells. The application of amber codon suppression in living cells warrants some discussion (cf., Figure 21a). First, protein production for ensemble spectroscopic experiments routinely demands a significant amount of sample. For example, the FTIR studies on azF-tagged rhodopsin have taken advantage of the fact that purified functional rhodopsin harboring uaas can be obtained at submilligram scale from mammalian cell culture.476,477 Other uaa-tagged GPCRs had to be analyzed by more sensitive assays like photo-cross-linking and fluorescence.206,403 As stated earlier, prokaryotic cells are generally unable to express correctly folded and post-translationally modified GPCRs. Thus, the major bottleneck was the lack of efficient eukaryotic expression systems for amber codon suppression, which has limited the applicability of a wide range of uaas to bacterially expressed proteins. The newly developed stable cell lines with a tRNA/aaRS pair for amber codon suppression452 may prove advantageous for this purpose. Second, 23.5% of the endogenous genes also use the amber stop codon.419 The consequences of off-target amber codon suppression remain understudied. The viability of uaa-tagged cells and animals suggests that amber codon suppression does not cause severe cytotoxicity. However, any interference with the cellular signaling network cannot be excluded. Third, amber codon suppression for an overexpressed POI is incomplete (typical efficiency for a single mutation: 5−20% using the tyrosyl tRNA/aaRS pairs) due to the competition of the eRF1/eRF3 complex, yielding truncated polypeptides. Although the truncated peptides are likely to be misfolded and then degraded by the proteasome, the efficiency of such cellular quality-control machinery and the cellular consequences are unclear. Last, the nonspecific substrate usage of the tRNA/aaRS pair may produce full-length protein even without exogenous uaas. In the majority of the published reports, the “leakiness” of a tRNA/aaRS pair is typically determined by Western blot, whose sensitivity depends on the affinity of the primary antibody and the sample processing procedure. However, the absence of nonspecifically expressed protein in the Western blot does not preclude any detectable activity in cell-based assays, particularly in highly sensitive assays (e.g., patch clamp or luciferase reporters). Also, when the amber codon is positioned close to the N-terminus, internal translation

4.13. Fluorogenic Labeling Reactions

Fluorogenic reactions can be particularly useful for achieving high contrast in the complex cellular environment.557−560 There are two popular strategies for designing fluorogenic probes. In the first strategy, the reactive moiety serves as the quencher for the linked dye. Conjugation with the cognate reactive handle on the POI destroys its quenching effect (Figure 24a). The first example for fluorogenic click reaction involved azido-hydroxycoumarin,561 followed by a variety of azido-based fluorogenic dyes.557 Later tetrazine was found to fulfill the fluorogenic criterion as well.562−564 The azide group functions as a PET quencher for coumarins,561 anthracene derivatives,565 xanthene derivatives,566,567 etc. The 1,3-dipolar cycloaddition of an azide and a terminal or strained alkyne converts the azide into a triazole ring, resulting in the loss of PET quenching (Figure 24b). Similarly, the quenching effect of tetrazine group is deactivated upon reacting with strained alkenes or alkynes (Figure 24c).552,562,568 In the second strategy, the quencher and the dye are connected by a cleavable linker (Figure 24e). Upon reaction, the concomitant release of the quencher unmasks the fluorescence emission. The modular nature of SNAP-tag (Figure 24f) and TMP-tag (Figure 24g) substrates was harnessed to design the second type of fluorogenic probes.311,569,570 The quencher is attached to the leaving moiety so that only the fluorophore ends up attached to the receptor. 4.14. Choosing the Right Labeling Method To Understand the Biochemistry and Cell Biology of GPCRs

4.14.1. Tracking GPCR Conformational Change. Various spectroscopic methods have been applied to track the conformational change in the course of GPCR activation. The probes, together with the linker to the protein, should be as small as possible to report the movement of the polypeptide backbone faithfully. On the other hand, a longer linker may facilitate probe reorientation and reduce orientational artifacts in FRET-based assays. The probes should also give strong signals so that receptor expression is less likely to be a hurdle. In the past, such probes were typically attached to the receptor through chemistries targeting cysteine thiols. The development of genetically encoded unnatural amino acids has the potential of overcoming this classic approach. The uaas can be incorporated into the TM region of GPCRs that is not accessible by most labeling chemistries. If the probe is only a few atoms in size and can be incorporated as part of the uaas, the linker length between the probe and the protein backbone can be dramatically reduced. As for the probes that cannot be genetically encoded, bioorthogonal labeling of uaa-tagged GPCRs may offer a general solution for site-specifically attaching them to GPCRs. As compared to chemistries targeting cysteine, bioorthogonal labeling targeting uaas benefits from its greater freedom in choosing the site of labeling. The fluorogenic bioorthogonal labeling strategy may ultimately enable single-molecule fluorescence studies of receptor conformational changes in the cell membrane. AE

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Another approach for probing conformational change is to utilize conformational-specific biomolecules, such as a ligand, an antibody, or a nanobody. Preparing such reagents is nontrivial, nonetheless. In the existing literature, the GFPnanobody biosensor198 represents an interesting case. Expressing the nanobody biosensor in the cellular milieu enabled one to overcome the barrier of the plasma membrane, which may open new possibilities for tracking GPCR conformational changes in organelles. 4.14.2. Trafficking and Internalization. Any method resulting in stable and bright labeling of a GPCR is theoretically useful in tracking the cellular localization of the receptor. The most popular strategies are based either on an epitope-specific antibody or on a fluorescent protein fusion. For example, a quantum dot-labeled high-affinity antibody was used to visualize the internalization and endosomal trafficking of epitope-tagged serotonin receptors.571 The question is whether such modification may alter the native behavior of the receptors. Antibodies, fluorescent proteins, and self-labeling proteins are all not much smaller in size than GPCRs (Figure 25), which may lead to interferences with receptor function. In fact, alteration of receptor mobility caused by fluorescent protein tagging has been reported.572 Ligand-directed labeling has also been successfully applied to monitor the internalization of membrane proteins.224 However, the prerequisite of this approach is that the ligand itself will not induce receptor internalization. By comparison, bioorthogonal chemistry targeting uaa-tagged GPCRs would produce minimal modification of receptors. It would be of general interest to evaluate whether this strategy is suitable for tracking the cellular localization of GPCRs. 4.14.3. Oligomerization. As described in section 3.5, much remains to be elucidated about GPCR oligomerization. Figure 26 summarizes the methods for detecting GPCR oligomerization within the scope of this Review. Most of them utilize FRET between fluorescent probes attached to monomeric receptors (Figure 26a−f). As compared to the methods based on fluorescent antibodies or protein tags, using fluorescent ligands for imaging GPCR oligomers (Figure 26b,c) has two important advantages. First, it is possible to image endogenously expressed GPCRs in native tissue because there is no need to overexpress a modified receptor expression construct. Second, fluorescent ligands are smaller than antibodies or protein tags. However, the use of fluorescent ligands, particularly in the case of fluorescent bivalent ligands, is limited by the availability of such reagents. Proximitydependent enzymatic labeling methods, like ID-PRIME or BioID, report protein interactions based on the physical proximity of binding partners (Figure 26g). While there is but one example in the published literature that applied IDPRIME to detect GPCR oligomerization,344 this strategy has a great potential for understanding GPCR signaling networks. Apart from fluorescence techniques, the degree of oligomerization can be analyzed by chemical cross-linking/mass spectrometry, raising the possibility of profiling GPCR oligomerization.

Figure 25. Comparing the sizes of GPCR, proteins tags, and fluorescent reporters. All of the molecules, as well as the quantum dot, are shown in scale. The crystal structures of (a) an immunoglobulin G (IgG, PDB: 1IGT),668 (b) a representative GPCR rhodopsin (Rho, PDB: 1U19),109 (d) GFP (PDB: 1GFL),669 (e) SNAP-/CLIP-tag (PDB: 3KZY), (g) Halo-tag (PDB: 4KAA), (h) TMP-tag (PDB: 1DR7), and (i) Renilla luciferase (RLuc, PDB: 2PSD)670 were prepared using VMD.671 The IgG molecule and its Fab and Fc regions illustrate the size of typical labels for immunofluorescence. The chromophore of GFP is highlighted in orange, and the active sites for the SNAP-/CLIP-tag (C145), Halo-tag (D106), and TMP-tag (L28C) are in red. The molecular model for Alexa647 (c) was generated using Schrödinger Maestro. The transmission electron micrograph (f) shows the structure of a 12 nm (CdSe)ZnS core−shell quantum dot with far red emission, similar to Alexa647. Electron micrograph courtesy of Andrew M. Smith. Reproduced with permission from ref 672. Copyright 2010 American Chemical Society.

be labeled with a fluorescent dye precisely at a 1:1 molar ratio (and not just an average dye/protein ratio of 1:1) using the methods described in the previous section. Despite a long list of papers in which over 45 different GPCRs have been investigated, the majority of studies were focused on tracking GPCR diffusion in the cell membrane, largely due to the longstanding limitation in site-specific labeling of the receptors. With the recent introduction of site-specific labels via SNAP-tag technology or unnatural amino acids (Tables 3 and 4), GPCRs can be selectively labeled in the cellular complexity. Consequently, important questions related to GPCR biology, such as conformational dynamics, ligand-binding dynamics, or mesoscale organization of GPCRs in the cell membrane, can be tackled in a meaningful way.

5. APPLICATION OF SINGLE-MOLECULE METHODS TO GPCRs Single-molecule imaging is ideally suited to decipher the dynamic and heterogeneous behaviors of GPCRs. In this section, we review the extensive literature reports in which single GPCRs were observed (Table 1). To conduct singlemolecule fluorescence imaging of GPCRs, each receptor has to AF

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Figure 26. Detecting GPCR oligomerization in living cells. Most detection schemes are based on energy transfer between (a) fluorescently labeled antibodies, (b) two fluorescent ligands, (c) a fluorescent bivalent ligand, (d) fluorescent proteins, (e) luciferase and a fluorescent protein, and (f) fluorophores conjugated to the orthogonal SNAP-tag and CLIP-tag. GPCR oligomerization can also be probed by proximity-dependent labeling, for example, (g) Interaction-Dependent Probe Incorporation Mediated by Enzymes (ID-PRIME).

5.1. Mobility, Oligomerization, and Stoichiometry

of GPCRs does not seem to be a general requirement for ligand recognition or signaling, but it is speculated as a mechanism for the cell to modulate receptor mobility at the cell surface, receptor intracellular trafficking, or receptor signaling functions. Current models describe the plasma membrane as a complex dynamic heterogeneous distribution of lipids and proteins in which signaling from cell surface receptors is often highly compartmentalized, with receptors existing in signaling microdomains such as caveolae or lipid rafts whose organization is mediated by specific protein−protein or protein−lipid interactions. In this context, the lateral mobility of receptors is a key parameter describing how they might move in and out of such microdomains and encounter other identical or different receptors to form transient or stable dimers or oligomers. The lateral mobility of GPCRs was initially investigated by fluorescence recovery after photobleaching (FRAP), using receptors tagged with a fluorescent protein or labeled with a fluorescent ligand. In a FRAP experiment, a defined area of the

Many membrane receptors function as homo- or heterodimers, or even as higher order oligomers, and oligomerization confers unique properties that monomers lack. For example, each monomer may contribute to the formation of the ligand binding site or recruiting intracellular adapter proteins. For a long time it was thought that GPCRs were an exception among membrane receptors by functioning as a monomer. Singlemolecule colocalization imaging and step-photobleaching analysis provided unequivocal evidence that single monomeric β2AR and μOR molecules incorporated into membrane-mimic high-density lipoprotein particles were capable of binding and activating their G protein.144,573 Monomeric rhodopsin in solution was shown to activate its G protein transducin as well.147 Nonetheless, evidence has accumulated in parallel that GPCRs are also capable of forming dimers, although it is still far from clear when and where this process takes place under physiological conditions.151,574 Dimerization or oligomerization AG

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technique

AH

anti-FLAG-antimouse-F (ab′)2-QD anti-FLAG-antimouse-F (ab′)2-QD agonist-bodipy630 antagonist-bodipy630, A3AR-GFP α1AR-CFP, Gaq-citrine

SPT

SPT

mobility

SPT fs-XR AFM

AFM AFM

AFM

AFM AFM

stability stability

stability

stability stability

α2AAR AT1R bR

β2AR

β1AR

SPT

mobility

SMLM

secondary antibody-Cy2 or Cy3 secondary antibodyAlexa Fluor 488 β2AR-mEos2 fusion

NSOM

distribution

SNAP-Alexa Fluor 647

SPT

B2R-GFP

mobility, signaling mobility, oligomers oligomers

FCS

FCS

SPT

mobility, oligomers mobility structure conformation

FCS FCS

C-terminal fusion to GFP or YFP α2AAR-SNAP

agonists-bodipy630 A2AAR-YFP

fusion to GFP, YFP, mCherry antagonist-bodipy630; A1AR-Topaz fusion agonist-bodipy630 A1AR-YFP

FCS FCS

FCS FCS

α1bAR

B2R

labeling antibody-QD antiGFP-biotinstreptavidin-QD655 C-terminal fusion to GFP or YFP

binding, mobility mobility mobility, oligomers mobility mobility, oligomers mobility

FCS

fs-XR FCS

FCS

SPT SPT

mobility mobility, oligomers mobility

A3R

A2AR

A1R

5-HT2BR 5-HT2CR

mobility, oligomers structure mobility

5-HT2AR

question

trafficking mobility

5-HT1AR 5-HT1BR

receptorb

Table 1. Published Single-Molecule Studies on GPCRsa summary

ref

157 626

visualization of ∼140 nm clusters of β1AR and β2AR in live mouse cells unstimulated β1AR and β2AR are confined due to interactions of the C-terminus with PDZ domain and A-kinase anchoring proteins but not caveolae application of PALM to estimate the molecular density in HeLa cells

583

625

623 624

622

620 621

294

618 602 619

607

617

615 616

experimental protocol to study receptor mobility structure with bound antagonist demonstration that pulling on bR in nanodiscs and in native purple membranes yields the same intermediates, demonstrating the usefulness of nanodiscs polypeptide loops potentially act as a barrier to unfolding and contribute significantly to the structural stability of BR individual structural segments of rhodopsin and bR have different properties; a core of rigid structural segments was observed in rhodopsin but not in bR point mutations can reshape the free energy landscape of a membrane protein and force single proteins to populate certain unfolding pathways over others mutations in extracellular Glu and Gln affect unfolding energy landscape characterization of inter- and intramolecular interactions stabilizing structural segments of bR assembled into trimers and dimers, and monomers association of B2R with Gq assessed by FCS; a portion of the receptors diffuses with a diffusion coefficient consistent with dimers or oligomers; no FCCS agonist stimulation increases the lateral mobility of GABAB receptors, but not of β1-/β2ARs

diffusion of Gaq in supported bilayers, either as monomers or as heterotrimers; heterotrimers are more immobile and partition into microdomains near GPCRs brightness analysis shows that the 6 tested receptors diffuse as homodimers in the cell membrane

two populations of diffusing A3AR exist determination of diffusion rate of A3AR dimers, and of the off-rate of the antagonist with and without an allosteric modulator

614

613

agonist-activated receptor is confined when C-terminal cysteine is palmitoylated, explaining the restricted collision coupling to Gs two diffusion states of A2AR in neurons

612 611

610 611

81

608 609

607

571 606

proof-of-concept that these ligands can be used for FCS determination of the diffusion rate of A1AR homodimers, A2AR homodimers, and A1AR−A2AAR heterodimers

uantification of receptor−ligand binding by monitoring the amplitude of the diffusing fraction corresponding to this population in live CHO cells demonstration that fluorescent ligands can be used to monitor diffusion of A1AR in live CHO cells determination of the diffusion rate of A1AR homodimers, A2AR homodimers, and A1AR−A2AAR heterodimers

XFEL structure corresponds to a more accurate room-temperature structure evidence for dimers, without monomers or tetramers

brightness analysis shows that the 6 tested receptors diffuse as homodimers in the cell membrane

internalization and endosomal trafficking of single groups of receptors recruitment of the receptor from soma to dendrites follows an unusual route via vesicle aggregates; lateral diffusion slowed at synapses

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AI

question

EM SM TIRF, EM AFM

oligomers

structure stoichiometry stability

EP2

mobility, oligomers mobility

SPT

FCS

SMLM

signaling

D1R

colocalization SMLM SM TIRF SMLM

oligomers distribution binding signaling

mCB2R CCR5

SPT

SPT localization SPT

mobility endocytosis mobility

mobility

SM TIRF colocalization FCS, TIRF SPT

stability mobility, oligomers conformation oligomers mobility mobility

AFM FCS

NSOM

distribution distribution structure mobility mobility

NSOM

cAR1-Halo-QD cAR1-eYFP cAR1-eYFP, cAR1-Halo. TMR secondary antibodyQD655 SNAP-505, Halo-TMR mAb-ATTO655 CCL3-Alexa Fluor 647 GFP-nanobodyATTO655 GFP-nanobodyATTO655 C-terminal fusion to GFP or YFP HAtag-AntiHA-QD655

C-terminal fusion to GFP or YFP β2AR-Cy3 SNAP-505, Halo-TMR C5a-YFP cAR1-Halo-TMR

arterenol-Alexa Fluor 532 secondary antibody-Cy2 or Cy3 negative stain β2AR-TMR β2AR-mEos2 β2AR-mEos2 negative stain SNAP-tag secondary antibodyAlexa Fluor 555 β2AR-Venus, β2ARGFP, β2AR-eYFP negative stain β2AR-Cy3, b2AR-Cy5

FCS

labeling TMR TMR SNAP-Alexa Fluor 647

technique

SM confocal SM confocal SPT

EM FCS SMLM SMLM EM SPT SPT

structure

conformation conformation mobility, oligomers mobility, binding oligomers

CB1R

C5a cAR1

receptorb

Table 1. continued

description of the tracking method and labeling protocols

brightness analysis shows that the 6 tested receptors diffuse as homodimers in the cell membrane

303

607

588

305 586 39 70

GABAB2 homodimerizes, β2AR and mCR2 do not dSTORM microscopy of CCR5 in filopodia of CHO cells proof-of-concept ligand-binding assay on CCR5 dSTORM microscopy reveals arrestin3 clustering after stimulation of CCR5 dSTORM microscopy reveals arrestin2 clustering after stimulation of CCR5

633

306 632 303

388 305 631 302

630 607

598 144 629

agonist binding leads to decreased mobility and internalization; decreased mobility key to desensitization

Cy3 used as a probe, fluctuations between two intensity states, active and inactive GABAB2 homodimerizes, β2AR and mCR2 do not kinetics of receptor trapping into clathrin-coated pits cAR1 diffusion is related to microtubule stability but not actin filaments in the amoeba Dictyostelium discoideum; two diffusing populations observed proof of principle of labeling strategy; Halo-QD and Halo-TMR have the same diffusion properties phosphorylation-dependent internalization of cAR1 description of the tracking method and labeling protocols

the Ga-helical domain undergoes a nucleotide- dependent transition from a flexible to a conformationally stabilized state demonstration that β2AR is monomeric in nanodiscs ligand binding induces weak interactions instead of strong localized ones; however, interactions established upon ligand binding are sufficient to change conformational, energetic, kinetic, and mechanical properties of structural segments of β2AR cholesterol increases the kinetic, energetic, and mechanical stability of β2AR brightness analysis shows that the 6 tested receptors diffuse as homodimers in the cell membrane

582

627 387 584 585 599 628 626

157

visualization of ∼140 nm clusters of β1AR and β2AR in live mouse cells description of the methodology observation of conformational substates β2AR partially clusters in cardiomyocytes, but not in other cell lines review (does not contain much information) structure of the receptor−arrestin complex protocol unstimulated β1AR and β2AR are confined due to interactions of the C-terminus with PDZ domain and A-kinase anchoring proteins but not caveolae larger size oligomers observed with GFP and eYFP, whereas size stays constant with Venus upon addition of agonist

594

membrane dynamics and internalization of β2AR in live hippocampal neurons; measurement of KD and kon

ref 27 590 294

summary first observation of the conformational dynamics of a single GPCR follow-up with more experimental details and inverse agonist agonist stimulation increases the lateral mobility of GABAB receptors, but not of β1-/β2ARs

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SMLM

SPT

mobility

AJ

P2R PAR1

OR17-40

OR5

NPY1R, NPY2R μOR

NK1R

M2R

M1R

LHR

H1R

mobility, oligomers mobility mobility, trafficking mobility binding

FCS

mobility, oligomers mobility, oligomers mobility, signaling mobility mobility, oligomers mobility mobility stoichiometry mobility

labeling

receptors insert into tethered bilayers and aggregate high constitutive activity observed, membrane diffusion and trafficking into endosomes followed

VSVtag-antiVSV-Cy5 OR17-40-GFP

SPT AFM

SPT ATP-QD

receptors insert into tethered bilayers and aggregate

FCS, TIRF

demonstration of labeling method, endocytosis, trafficking, recycling demonstration of method to determine the ligand-binding free-energy landscape

observation of confined diffusion observation of rapid confined diffusion and slow long-range diffusion reconstituted μOR is monomeric; one ligand binds per receptor contrary to Daumas et al.,641 they find rapid hop diffusion

antibody-gold antibody-gold μOR-YFP; agonist-Cy3 antibody-golf, μORmGFP VSVtag-antiVSV-Cy5

SPT SPT TIRF SPT

proof-of-concept labeling, heterogeneous diffusion observed NPY changes mobility and these changes reflect on event related to arrestin recruitment and endocytosis

receptor mobility decreases during the first second after binding of the ligand due to increased interactions with cellular structures

brightness analysis shows that the 6 tested receptors diffuse as homodimers in the cell membrane

brightness analysis shows that the 6 tested receptors diffuse as homodimers in the cell membrane

mobility, clustering, and dimerization kinetics of M1R in CHO cells, randomly distributed with 30% in dimers at any time

super-resolution imaging of functionally asymmetric oligomers reveals diverse functional and structural organizations and the ability to alter signal responses binding of gonadotropin confines the receptor to small domains, maybe rafts

receptor activation increases diffusion; scaffolding protein Homer favors confinement into Homer-mGluR5 clusters after a certain lag time, mGluR5 undergoes directed rearward transport in an actin-dependent way at the surface of neuronal growth cones diffusion of the receptor in the cell membrane

the ligand binding domain exists in three conformations, and orthosteric ligands drive changes between these conformations, leading to dimer interface remodeling mGluR3 has a more stable active state than GluR2 and can be activated by Ca2+

open and closed states of GCGR revealed kinetics of reorientation of extracellular loops; receptors oscillate between a resting and an active conformation on submillisecond time scale

50 647

645 646

644

641 642 573 643

639 640

638

607

607

578

637

587

635 636 593

592

592

600 295

305 628 634

294

agonist stimulation increases the mobility of GABAB receptors, but not of β1-/β2ARs GABAB2 homodimerizes, b2AR and mCR2 do not protocol two populations of GALR observed

304

description of method of observation of dimer lifetimes

ref 579

summary characterization of monomer−dimer equilibrium, unchanged by ligand

NK1R-ACP-CoA-Cy5 receptor-GFP (BiFC)

H1R-YFP, antagonistbodipy630 antiFLAG-Cage500 or Cage552 anti-FLAG-gold (40 nm gold) antagonist-Cy3B, antagonist-AF488 C-terminal fusion to GFP or YFP C-terminal fusion to GFP or YFP NK1R-EGFP

uranyl formate SNAP-mGluR2mGluR2-SNAP SNAP-mGluR2mGluR2-CLIP SNAP-mGluR3mGluR2-CLIP mGluR5-myc-GFP

ligand-Alexa Fluor 594; FPR-YFP (BiFC) Fab-fluorophore, Halotag, ACP-tag SNAP-Alexa Fluor 647 on GABAB1 SNAP-505, Halo-TMR SNAP-tag galanin-rhodamine

SPT FCS

SPT

FCS

SPT

dimers

SPT SPT FCS

mobility mobility binding, mobility oligomers

mGluR5

smFRET

conformation

smFRET

EM smFRET

conformation conformation

conformation

colocalization SPT FCS

mobility, oligomers oligomers mobility mobility

SPT

SPT

oligomers

technique

SPT

question

oligomers

mGluR3

GABAB1GABAB2 GABAB2 GABAB GALR1, GALR2 GCGR mGluR2

FPR

receptorb

Table 1. continued

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AK

b

a

SPT

TIRF AFM, EM, review AFM AFM AFM AFM AFM

AFM

mobility

mobility

imaging oligomers

stability

FCS, FCCS

SPT

oligomers

mobility

SPT SPT

mobility mobility

EM

structure

AFM

AFM

stability

binding

AFM AFM

stability stability

oligomers oligomers stability stability stability

SPT

structure structure mobility

technique

AFM SPT review EM fs-XR SPT

question

binding mobility

SNAP-tag biotin-tag-streptavidinAlexa Fluor 647 SST-FITC, SSTTexasRed biotin-tag-streptavidinAlexa Fluor 647

negative stain

GTa-peptideATTO647N GTa-peptideATTO647N GTa-peptideATTO647N azF+SpAAC negative stain

gold clusters

ACP-QD

labeling

summary

smoothened and SSTR3 move predominantly by diffusion in cilia; attachment to intraflagellar transport trains is transient and stochastic

homo- and hetero-oligomers are occupied by two ligands

binding events disrupt the primarily diffusive motion of smoothened in cilia smoothened and SSTR3 move predominantly by diffusion in cilia; attachment to intraflagellar transport trains is transient and stochastic

demonstration that rhodopsin forms dimers in native discs rhodopsin and opsin oligomerize in native disc membranes importance of one disulfide bridge for overall stability stabilizing interactions stabilizing mouse and bovine rhodopsin are conserved compared to dark state wild-type rhodopsin, the G90D mutation decreased energetic stability and increased mechanical rigidity of most structural regions in the dark state mutant receptor individual structural segments of rhodopsin and bR have different properties; a core of rigid structural segments was observed in rhodopsin but not in bR Zn2+ increases the stability of most structural segments the absence of palmitoylation in rhodopsin, therefore, destabilizes the molecular interactions formed at the carboxyl terminal end of the receptor, which appears to hinder the activation of transducin by light-activated rhodopsin compared to dark-state rhodopsin, the structural segments stabilizing opsin showed higher interaction strengths and mechanical rigidities and lower conformational variability, lifetimes, and free energies pentameric assembly of the rhodopsin-Gt complex in which a photoactivated rhodopsin dimer serves as a platform for binding the Gt heterotrimer transducer binding establishes localized interactions to tune sensory rhodopsin II

demonstration of labeling, antibody-mediated capturing, and observation on glass surface review on AFM and EM measurements demonstrating oligomeric structure in native membranes

interactions of Gt with rhodopsin are favored at the rim of the membrane

binding of G to rhodopsin, mobility of activated rhodopsin

AFM mapping of two different ligand-binding events using a chemically bifunctionalized AFM tip demonstration of the labeling method fluorescence spectroscopy of rhodopsins demonstration of incorporation of functional rhodopsin into NABBs; EM images demonstrating stoichiometry and orientation active form of rhodopsin bound to arrestin slow and less restricted G diffusion in the active state of rhodopsin

ref

663

664

662 663

661

660

659

657 658

621

155 156 654 655 656

509 580

653

652

648 649 650 145 114 651

This table covers the single-molecule studies on GPCRs to the best of our knowledge. However, we do not guarantee that the list is completely comprehensive and apologize for any unwanted omission. This table only shows the abbreviations of GPCRs. Please refer to Table 2 for their full names.

SSTR1, SSTR5 SSTR3

sensory rhodopsin II smoothened

PTHR rhodopsin

receptorb

Table 1. continued

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membrane diffuse into the bleached area. In addition to FRAP, FCS has also been used to investigate the mobility of over 15 different GPCRs in the membrane of live cells.575 However, both FRAP and FCS suffer from the fact that they only yield average diffusion times and can therefore hardly account for local heterogeneities in the cell membrane or provide information on receptor stoichiometry. The single-molecule methods commonly used to study membrane receptor oligomerization include single-molecule photobleaching, smFRET, SMLM, and SPT.576 Single fluorescent molecule video imaging is certainly the most suitable method for visualizing dynamic molecular interactions in live cells and characterizing the diffusion of GPCRs: it not only reports on the variations of the diffusion of fluorescently labeled receptors over time and space, but also tells whether diffusing receptors oligomerize and, if they do, for how long the interaction lasts before an oligomer dissociates into monomers.151,577 The first single-molecule demonstration for the transient dimerization of GPCR in living cells was achieved by Hern et al. utilizing an antagonist of the M1 muscarinic receptor derivatized with Alexa Fluor 488 or Cy3B.578 By tracking individual antagonist-bound receptors in the two channels corresponding to the two fluorophores, the authors were able to demonstrate transient correlated motion of two receptor molecules heterologously expressed in CHO cells, indicating transient dimer formation. They found that the dimers quickly dissociated again into monomers, with an average time constant of 0.7 s (at 23 °C). Whether the M1 muscarinic receptor formed dimers in the absence of the antagonist at physiological temperatures in a more relevant cell line remains unknown. Kasai et al. later fully characterized the monomer−dimer equilibrium of the N-formyl-peptide receptor (FPR) in CHO cells at 37 °C using a fluorescently labeled agonist and an FPRYFP fusion.579 They observed no change in the monomer− dimer equilibrium upon ligand binding, with a typical association time of ∼150 ms and a dissociation time of ∼90 ms. Calebiro et al. observed transient homodimers with lifetimes of about 4 s at 20 °C for β1AR and β2AR labeled via a SNAPtag in CHO cells.294 Similarly, ligand binding did not alter the equilibrium or affect the mobility of the receptors. Interestingly, β2AR seemed to have a higher tendency to form dimers than β1AR at a given expression level. The authors suggest that this apparent difference in converting a collision into a stable interaction might arise from other proteins capable of interfering with dimerization, or from localizations in different microdomains that lead to different effective densities of the two receptors. Calebiro et al. also characterized GABAB receptors for which there is strong evidence for the dimerization between a GABAB1 and a GABAB2 subunit under physiologically relevant conditions. They found GABAB receptors to be in equilibrium between heterodimers and higher-order oligomers, with a preference for tetramers and octamers. With this prototypic class C receptor, an increase in the lateral mobility was observed after agonist binding, suggesting that the ligand can modulate interactions between the receptor and the actin cytoskeleton. All of these studies highlight the dynamic nature of receptor−receptor interactions and suggest that transient dimer or oligomer formation might be a general mechanism for GPCRs, at least in these artificial cellular backgrounds. Kasai et al. even proposed that dynamic homodimers must be crucial for some GPCR functions, which remains to be verified, and

Table 2. Abbreviations of GPCRs GPCR bR 5-HT(x)R A(x)R αxAR βxAR AT1R C5αR cAR1 CB1R CCKAR CCR5 mCBR1 D1R EP2 FPR GABA(x) GCGR mGluR(x) H1R LHR MxR NK(x)R NPY(x)R μOR OR(x) P2R PAR1 PTHR SSTR(x)

G protein-coupled receptor bacteriorhodopsin (not a GPCR, but its heptahelical transmembrane domain is structurally related to GPCRs) 5-hydroxytryptamine (serotonin) receptor subtype x adenosine receptor subtype x αx-adrenergic receptor βx-adrenergic receptor angiotensin II receptor type 1 complement component 5a receptor 1 cAMP receptor 1 cannabinoid type 1 cholecystokinin receptor type A chemokine CCR5 receptor mouse cannabinoid receptor 2 dopamine receptor type 1 prostaglandin E2 receptor N-formyl peptide receptor γ-aminobutyric acid receptor type x glucagon receptor metabotropic glutamate receptor type x histamine receptor type 1 luteinizing hormone receptor muscarinic acetyl choline receptor subtype M-x neurokinin-(x) receptor neuropeptide Y receptor type x μ-opioid receptor olfactory receptor family x P2 purinergic receptor protease-activated receptor-1 parathyroid hormone receptor somatostatin receptor type x

Table 3. Abbreviations of Unnatural Amino Acids uaa 5-OH-Trp AcrK Aladan AlkK AzK Anap AcF azF BCNK BzF (BODIPYFL)K CpK CoK1 CoK2 DansA Ffact HceG NBD-Dap NorK1 NorK2 TCOK ThiPK

unnatural amino acid 5-hydroxyl-L-tryptophan Nε-acryloyl-L-lysine β-[6′-(N,N-dimethyl)amino-2′-naphthoyl]-L-alanine Nε-[(2-propynyloxy)carbonyl]-L-lysine Nε-[(2-azidoethoxy)carbonyl]-L-lysine 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid p-acetyl-L-phenylalanine p-azido-L-phenylalanine Nε-[bicyclo[6.1.0]non-4-yn-9-methyloxy)carbonyl]-L-lysine p-benzoyl-L-phenylalanine boron-dipyrromethene-L-lysine Nε-(1-methylcycloprop-2-enecarboxamido)-L-lysine Nε-[(cyclooct-2-yn-1-yloxy)carbonyl]-L-lysine Nε-(2-(cyclooct-2-yn-1-yloxy)ethyl) carbonyl-L-lysine 2-[[5-(dimethylamino)naphthalene-1-yl]sulfonylamino] propanoic acid (dansylalanine) p-fluoroacetyl-L-phenylalanine L-(7-hydroxycoumarin-4-yl)ethylglycine 3-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-2,3diaminopropionic acid Nε-[(5-norbornene-2-yloxy)carbonyl]-L-lysine Nε-[(endonorborn-2-en-5-methyloxy)carbonyl]-L-lysine Nε-[(trans-cyclooctene-4-ol)carbonyl]-L-lysine Nε-thiaprolyl-L-lysine

cell membrane containing the labeled POI is photobleached. The fluorescence intensity then recovers over time because labeled molecules from neighboring regions of the cell AL

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Table 4. Other Abbreviations aaRS ABEL AFM ALEX APD APEX BG BRET BSA BTFA CFP cpGFP CuAAC cryo-EM DEER eDHFR EMCCD EPR FCS FGE FlAsH FP FRAP FRET FTIR GFP GPCR HDX-MS IC ID-PRIME ISC LDAI LDT MAPK mBB (or mBBr) ML

MSD NAMs NSOM PAMs PEG PET PI3K PMMA PMT POE POI PROXYL PSF Pyl ReAsH RhoBo RLuc SAM SCAM sCMOS Sel SILAC smFRET SMLM SpAAC SPIEDAC

aminoacyl tRNA synthetase anti-Brownian electrokinetic atomic force microscopy alternating laser excitation avalanche photodiode ascorbate peroxidase benzylguanine bioluminescence energy transfer bovine serum albumin 3-bromo-1,1,1-trifluoroacetone cyan fluorescent protein circular permuted GFP copper-catalyzed azide−alkyne [3+2] cycloaddition cryo-electron microscopy double electron−electron resonance E. coli dihydrofolate reductase electron-multiplying charge coupled device electron paramagnetic resonance fluorescence correlation spectroscopy formylglycine generating enzyme fluorescein arsenical hairpin binder fluorescent protein fluorescence recovery after photobleaching Förster resonance energy transfer Fourier transformation infrared green fluorescent protein G protein-coupled receptor hydrogen−deuterium exchange mass spectrometry internal conversion interaction-dependent probe incorporation mediated by enzymes intersystem crossing ligand-directed acyl imidazole chemistry ligand-directed tosyl chemistry mitogen-activated protein kinases monobromobimane

SPT TIRF TM TMP TMR Trp Tyr UVB VR YFP

mean squared displacement negative allosteric modulators near-field scanning optical microscopy positive allosteric modulators polyethylene glycol photoinduced electron transfer phosphatidylinositol-3 kinase poly(methyl methacrylate) photomultiplier tube polyoxyethylene protein of interest pyrrolidinyloxy (free radical) point spread function pyrrolysine resorufin arsenical hairpin binder rhodamine-based bisboronic acid Renilla luciferase silent allosteric modulators substituted-cysteine accessibility method scientific complementary metal-oxide-semiconductor selenocysteine stable isotope labeling by amino acids in cell culture single-molecule Förster energy transfer single-molecule localization microscopy strain-promoted azide−alkyne [3+2] cycloaddition strain-promoted inverse-electron-demand Diels−Alder cycloaddition single-particle tracking total-internal-reflection fluorescence transmembrane trimethoprim tetramethylrhodamine tryptophan tyrosine ultraviolet B vibrational relaxation yellow fluorescent protein

maleimide

performed by NSOM using receptors fused to a fluorescent protein. It was found that these receptors were organized in nanodomains with a diameter of ∼150 nm and did not reorganize upon agonist binding.157,582 NSOM is nonetheless particularly difficult to implement in living cells. More recently, SMLM was used to re-evaluate the molecular density of the β2AR in cardiomyocytes, HeLa cells, and CHO cells.583−585 It was found that the receptor indeed preassembled in clusters typically 100−200 nm in size in cardiomyocytes and that the distribution did not significantly change upon addition of ligands, corroborating the findings of the NSOM studies. However, no clustering was observed in HeLa or CHO cells, which was consistent with the findings of the tracking studies discussed earlier294 and with the fact that the β2AR is fully functional as a monomeric entity.144 The absence of significant clustering was further confirmed in CHO and HEK cells by colocalization analysis.305 Similarly, no significant cluster formation in CHO cells was found for the HIV entry coreceptor CCR5, another rhodopsin-like GPCR, although it did accumulate to high densities in the filopodia of these cells.586 On the other hand, the luteinizing hormone receptor (LHR) was shown to mostly form oligomers in HEK cells.587 In this case, however, thanks to an experimental localization

that downstream signaling through G proteins, kinases, or arrestins might be differentially induced by monomers and dimers.577 Single-molecule imaging would be suitable for obtaining a deeper understanding on these biologically relevant questions. 5.2. Membrane Organization beyond the Diffraction Limit

Determining the stoichiometry of higher order oligomers by traditional single-molecule imaging can become very difficult once the number of entities reaches a handful or more. Larger oligomeric assemblies of GPCRs were first described in the context of much debated atomic force microscopy images of rows of rhodopsin dimers in the retina.155,156,580 AFM is however not suitable for most GPCRs, whose expression level under physiological conditions is orders of magnitude lower than rhodopsin. Super-resolution imaging techniques have demonstrated their ability to provide relative or absolute quantitative information about protein copy numbers in oligomers and clusters.69,581 These methods have recently been applied to investigate the organization of GPCRs and arrestins in the cell membrane. Visualizing β1AR and β2AR clusters in cardiomyocytes, whose contraction is controlled by their activation, was first AM

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recording of the fluorescence intensity and lifetime, the authors found a range of discrete conformational states with dwell times of hundreds of milliseconds. The addition of a high affinity agonist increased the dwell times of these states. Millisecond fluctuations were also observed within these conformational states, suggesting that β2AR dynamics spanned a wide range of time scales. Counterintuitively, no large change in the conformational states or the interconversion dynamics occurred upon addition of an agonist, which would be expected from receptor activation. A plausible explanation was the absence of G protein in these experiments. Subsequent structural and NMR studies revealed that a G protein was indispensable for β2AR to reach the fully activated conformational state.221,394 In a recent study, the same β2AR construct was labeled at C265 with Cy3, incorporated into nanodiscs, and immobilized on a glass surface using streptavidin−biotin technology.388 Transitions between two distinct states with dwell times on the order of several seconds were observed in the single-molecule TIRF experiment. The states were assigned to inactive and active-like receptor conformations. Unliganded receptor molecules repeatedly switched between both conformations, leaning toward the inactive conformation. The addition of an agonist favored the active-like conformation, whereas binding of an inverse agonist shifted the conformational distribution toward the inactive conformation. The agonist also enhanced the frequency of activation events, while reducing the frequency of deactivation transitions. The inverse agonist, however, increased the frequency of deactivation events. Aided by molecular modeling, the authors suggested that their ability to observe conformational transitions with Cy3 might have originated in changes in the local confinement of the dye: the inactive conformation was predicted to confine the dye between TM3, 4, and 5, whereas the dye was expected to be in a fully exposed solvent environment in the active state. Cy3 in its lowest excited state returns to the ground state without emission through trans-to-cis isomerization. In the active state of β2AR, the isomerization of Cy3 would be less impeded by the local environment, thereby leading to more efficient quenching of Cy3 fluorescence. This property of Cy3 has been exploited in a variety of studies to probe protein conformational change.591 Experiments relying on a single fluorescent reporter often suffer from an important limitation: signal fluctuation arising from changes in the local environment can barely be correlated to specific structural changes. By comparison, FRET between two fluorophores is more serviceable for interrogating GPCR conformational changes. Previously, FRET experiments on GPCRs have been impeded by the difficulty of attaching synthetic dyes to the receptors. The invention of SNAP- and CLIP-tags has greatly expanded the choices for fluorescent reporters. In a study examining the conformational change of SNAP- and CLIP-tagged metabotropic glutamate receptor (mGluR), which are known to be active only as dimers,295 the kinetics of the reorientation of the extracellular ligand-binding domain of freely diffusing purified receptor dimers were monitored by multiparameter fluorescence detection. In this case, while the observation time of single dimers was limited, the authors reported oscillations between a resting and an active conformation on a submillisecond time scale and showed that agonist efficacy could be correlated with the ability of the ligand to shift the conformational equilibrium toward the active state.

precision of less than 10 nm, analysis of the spatial arrangements of the molecular localization coupled to molecular modeling led the authors to postulate possible structural arrangements for trimers and tetramers. Another question that seems suitable for super-resolution methods to address is the stoichiometry and the duration of the interactions between GPCR and the intracellular adapter proteins. It was recently shown by SMLM imaging of arrestin2 and arrestin3 that different ligands binding to CCR5 induced differential formation of arrestin2 and arrestin3 clusters inside CHO cells.70,588 Whereas most ligands led to recruitment of both arrestins, one ligand caused clustering of arrestin2 but not of arrestin3. In these studies, arrestins were fused to GFP and detected with a fluorescent anti-GFP camel antibody (nanobody). Little is known about the specific physiological roles of arrestin2 and arrestin3, and further investigation with multicolor SMLM is likely to shed some light on the stoichiometry, the duration, and the localization of these interactions. 5.3. Conformational Dynamics

A major motivation for performing experiments on the singlemolecule level is to follow molecular dynamics of unsynchronized molecules in real time with the aim of observing rare or transient states hidden in ensemble measurements.589 Given the relevance of the conformational diversity of GPCRs with respect to their physiological function, it seems crucial to develop a better understanding of the dynamics of GPCR conformations. The development of a conformational assay on the singlemolecule level for GPCRs has nonetheless been hampered by the necessity of finding conditions in which a GPCR could be prepared in a form that is pure enough for single-molecule experiments, functional despite the absence of the cell membrane, and site-specifically labeled in a way that singlemolecule compatible dyes can report the conformational changes. For a long time, the only receptor to meet these very stringent criteria was β2AR, which was specifically labeled on an exposed cysteine residue (C265) in the third intracellular loop by thiol-reactive dyes in the minimal cysteine background.234 Various structurally related ligands were shown to induce different changes in fluorescence intensity of dyes including fluorescein, tetramethylrhodamine (TMR), or bimane specifically attached to C265.102,103,386 Because most of the tested ligands were agonists, such effects could not be unambiguously identified in a functional assay for β2AR that would behave similarly in response to the ligands. The changes in fluorescence intensity were ascribed to changes in the local environment of the dye, most likely related to polarity and confinement. Investigating the dynamics of these conformations was first attempted in an FCS-type of experiment, with single β2AR molecules diffusing through a confocal probe volume.387 The observed histogram of photon counts for single diffusing receptors was broad and displayed two maxima separated by a single bin, possibly corresponding to several conformational states. The major limitation of this study was that the receptors could be observed for no more than a few milliseconds, leaving no time to follow transitions in conformational states. The first observation of a single GPCR changing between its conformations came a decade later.27,590 An ABEL trap allowed β2AR molecules labeled with TMR to be observed for several seconds in detergent micelles. On the basis of simultaneous AN

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binding partners. Over the past decade, structural studies at the single-molecule level have been made possible by atomic force microscopy (AFM), cryo-electron microscopy (cryo-EM), femtosecond serial X-ray crystallography, as well as molecular modeling. Whereas AFM has been primarily used to investigate the mechanical stability of GPCRs in single-molecule force measurements,596 single-particle cryo-EM has revolutionized the field of structural biology in visualizing macromolecular structures, particularly large complexes that have resisted crystallization efforts, at resolutions that can compete against classic crystallography.597 Westfield et al. scrutinized the complex between an agonist, β2AR, and its G protein by cryo-EM.598 They found an overall architecture of the complex in good agreement with the crystal structure of the active-state ternary complex.119 Additionally, they reported the nucleotidedependent rearrangement of α-helical domain of the Gα subunit in the transition from a flexible state to a conformationally stabilized state. In another landmark paper, Shukla et al. examined the interaction between arrestin2 and β2AR by combining singleparticle cryo-EM with hydrogen−deuterium exchange mass spectrometry (HDX-MS), chemical cross-linking, and molecular modeling.599 The authors were able to present the first molecular model of the β2AR-arrestin2 signaling complex by docking the crystal structures of activated arrestin2 and of β2AR into the electron microscopy map densities with constraints provided by HDX-MS and cross-linking, yielding unprecedented insights into the overall architecture of a receptorarrestin complex. More recently, Yang et al. reported the structure of the glucagon receptor (GCGR) by using the same combination of techniques.600 This study revealed the open and closed states of GCGR, suggesting the glucagon binding through a conformational selection mechanism. Obtaining full-length structures at an atomic resolution of GCGR, a class B GPCR, which does not belong to the most abundant class-A (rhodopsin-like) GPCR family, has been very challenging due to the very dynamic nature of their extracellular domain and lack of highaffinity ligands to stabilize the receptor structure. The approach based on single-particle cryo-EM therefore appears very promising in complementing traditional crystallographic methods to provide insights into the structure and dynamics of GPCRs. In parallel to EM, breakthroughs in GPCR structural studies can be expected in the near future from pump−probe serial femtosecond crystallography, which uses the potential of X-ray free electron lasers for tracking the dynamics of light-triggered processes, such as rhodopsin activation.601 The static roomtemperature structures of an antagonist-bound angiotensin receptor602 and of rhodopsin bound to visual arrestin114 were solved by serial femtosecond X-ray crystallography. The possibility of obtaining meaningful static and dynamic structural information by molecular modeling and molecular dynamics simulations deserves to be credited here. With the ever increasing computational power of supercomputers, simulations spanning tens of microseconds of dynamics can be robustly performed to evaluate processes ranging from the activation mechanism of GPCRs to nucleotide exchange in heterotrimeric G proteins,603,604 demonstrating the potential of this type of simulations to serve as a “computational microscope”.605

In a follow-up study, Isacoff and co-workers harnessed the full power of single-molecule FRET to probe the conformational reorientation of the ligand-binding domain of mGluR homodimers.592 SNAP−mGluR and CLIP−mGluR were expressed in HEK293T cells, labeled a FRET donor and acceptor fluorophore, respectively, purified from the cell membrane, and captured on a glass surface in the scheme shown in Figure 6. Single dimers could be continuously observed for several tens of seconds. The authors were able to demonstrate that the ligand-binding domains interconverted between three conformations: resting, activated, and a shortlived intermediate state. Agonists induced transitions between these conformational states, with their efficacies being determined by occupancy of the active conformation. Overall, their results support a general mechanism for the activation of mGluR: agonist binding induces closure of the ligand-binding domain, followed by dimer interface reorientation. 5.4. Ligand Binding

Ligand binding to its GPCR is the key molecular event for triggering an intracellular signaling response. Nonetheless, literature reporting on ligand binding at the single molecule level is scarce. Such studies are highly desirable because they can provide quantitative information about the interaction between two molecules for deriving kinetic (kon and koff) and thermodynamic parameters (the equilibrium constant KD = koff/kon). A possible reason is that the accessible concentration range of single-molecule experiments typically falls into the picomolar to nanomolar range, whereas many biomolecular interactions require concentrations at least 100 times larger.18 Furthermore, a single-molecule binding assay demands both the receptor and the ligand to be simultaneously monitored; thus a fluorescent tag also needs to be attached to the ligand without impairing the receptor−ligand interaction. Whereas coupling a fluorescent tag to a peptide ligand without affecting its binding property seems reasonably easy if the peptide is large enough, this task proves much more complicated for small-molecule ligands.202,204,217 Fluorescent ligands have therefore often been used as an indirect way of labeling the receptor, for example, to follow the lateral mobility of the latter in the plasma membrane of living cells. Importantly, they increasingly replace dangerous radioactive tracers in affinity measurements based on integration of the total fluorescence response over a whole field of view.593 Despite all of these difficulties, FCS was used in one case to determine the binding constant of the fluorescently labeled agonist arterenol to β2AR in hippocampal neurons and in an epithelial cell line, with the KD determined to be 1.3 and 6.0 nM, respectively.594 The feasibility of a single-molecule binding assay on GPCRs was later evaluated for the receptor CCR5, whose natural ligands chemokines are ∼70 amino acid long peptides. The receptor was purified and embedded in a membrane tethered to the surface of a chip. Binding events of the fluorescently labeled chemokine CCL3 with CCR5, whose residence time lasted tens of minutes, could be monitored over the time course of hours.39 5.5. Structure and Stability

A detailed understanding of macromolecular processes and their dynamics requires the integration of information from a wide range of experimental and computational approaches covering different spatial and temporal regimes.595 Fluorescence methods can be limited when it comes to obtaining highresolution structural information for the receptor and its AO

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6. CONCLUSION AND PROSPECT Biochemical and biophysical characterization of GPCRs in the past two decades has led to a considerable appreciation of the dynamic and heterogeneous nature of GPCR signaling complex, which has given rise to the increasing popularity of single-molecule techniques in the GPCR field. The singlemolecule studies of GPCRs have benefitted from the interdisciplinary efforts in three areas. First, an intimate knowledge of the receptor biochemistry and cell biology necessarily underlies the success of any experiment. Thanks to the explosive development of molecular cloning and sequencing, experiments on heterologously expressed GPCRs in living cells have become more tractable. By comparison, studying purified GPCRs in a reconstituted system, despite its longer history, remains a major challenge. As a result, the literature of the biochemical studies on two prototypical GPCRs, rhodopsin and β2AR, appears to be disproportionally rich. Second, innovative single-molecule detection schemes and instrumentations have enabled a broader range of questions to be approached. Finally, the expanding chemical biology toolkits for protein modification have been harnessed to prepare labeled receptors that suit specific experimental designs. Labeling strategies aiming at facilitating single-molecule studies should involve receptors with minimal alterations of their native structure. The possibility of targeting GPCRs in native tissues with fluorescent ligands or ligand-directed labeling may overcome the limitations associated with heterologous expression systems, such as overexpression of receptor or cell type-specific background. Chemoenzymatic labeling provides a general approach for tagging GPCRs with different types of fluorescent labels. Previously, the transmembrane region and the ligand binding pockets of GPCRs were mostly studied by site-directed mutagenesis. The application of unnatural amino acids, combined with bioorthogonal labeling chemistries, may significantly advance spectroscopic and microscopic characterization of receptor conformational change.

photochemistry and ultrafast dynamics of biomolecules with Eric Vauthey at the University of Geneva (Ph.D. 2007) before moving into single-molecule spectroscopy and imaging as a postdoc with W. E. Moerner at Stanford University (2008−2010). In 2010, he started his independent research thanks to an Ambizione fellowship of the Swiss National Science Foundation at the Faculty of Medicine of the University of Geneva, his research focusing on the development and application of single-molecule tools for biology with an emphasis on the dynamics of G protein-coupled receptors. He has been a visiting assistant professor at The Rockefeller University in the group of Thomas P. Sakmar since October 2015. Thomas Huber graduated from the University of Munich in Medicine in 1995. He conduced his Ph.D. work with Klaus Beyer on NMR spectroscopy and molecular dynamics simulations of biological membranes in Martin Klingenberg’s Institute of Physical Biochemistry at the University of Munich (Ph.D. 1999). Huber then performed postdoctoral work with Michael F. Brown in the Department of Chemistry at the University of Arizona in Tucson to study lipid− protein interactions. He joined Thomas P. Sakmar’s laboratory at the Rockefeller University in 2002. Here, he studied receptor oligomerization and developed applications of unnatural amino acid mutagenesis in GPCR drug discovery. In 2013, he was appointed Research Assistant Professor. His research interests are in the area of Chemical and Quantitative Biology with a focus on single-molecule methods.

ACKNOWLEDGMENTS We acknowledge the generous support from a grant from the Robertson Foundation, the Crowley Family Fund, the Danica Foundation, and the NIH R01 EY012049 to T.H., as well as the Tri-Institutional Training Program in Chemical Biology for supporting H.T. This work was also generously supported by an International Research Alliance with Thue W. Schwartz at The Novo Nordisk Foundation Center for Basic Metabolic Research (http://www.metabol.ku.dk) through an unconditional grant from the Novo Nordisk Foundation to the University of Copenhagen. We also acknowledge Thomas P. Sakmar and the following members of his lab who contributed to this work: Kelly Daggett, Amy Grunbeck, Manija Kazmi, Adam Knepp, Saranga Naganathan, Minyoung Park, Sarmistha Ray-Saha, Carlos Rico, Pallavi Sachdev, Louise ValentinHansen, and Shixin Ye.

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected] Author Contributions †

H.T. and A.F. contributed equally to this work.

Notes

REFERENCES

The authors declare no competing financial interest.

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Biographies He Tian received her B.Sc. in Chemistry from Peking University, China, in 2009. She then moved to New York City to enroll in the TriInstitutional Ph.D. Program in Chemical Biology. She conducted her doctoral research under the supervision of Thomas P. Sakmar and Thomas Huber at the Rockefeller University and obtained her Ph.D. in 2015. Her dissertation focused on developing chemical biology tools for probing the structure−function relationship in G protein-coupled receptors. In 2016, she joined the research group led by Adam Cohen in the Department of Chemistry and Chemical Biology at Harvard University as a postdoctoral researcher. Her current research interest involves understanding the biophysical principles governing membrane proteins by protein engineering. Alexandre Fürstenberg studied chemistry and biochemistry at the Universities of Lausanne and Geneva in Switzerland. He specialized in AP

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DOI: 10.1021/acs.chemrev.6b00084 Chem. Rev. XXXX, XXX, XXX−XXX