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Hot spots for GPCR signaling: lessons from single-molecule microscopy
摘要： G protein-coupled receptors (GPCRs) are among the best-studied membrane receptors, mainly due to their central role in human physiology, involvement in disease and relevance as drug targets. Although biochemical and pharmacological studies have characterized the main steps in GPCR signaling, how GPCRs produce highly specific responses in our cells remains insufficiently understood. New developments in single-molecule microscopy have made it possible to study the protein–protein interactions at the basis of GPCR signaling in previously inconceivable detail. Using this approach, it was recently possible to follow individual receptors and G proteins as they diffuse, interact and signal on the surface of living cells. This has revealed hot spots on the plasma membrane, where receptors and G proteins undergo transient interactions to produce rapid and local signals. Overall, these recent findings reveal a high degree of dynamicity and complexity in signaling by GPCRs, which provides a new basis to understand how these important receptors produce specific effects and might pave the way to innovative pharmacological approaches.
关键词： single-molecule microscopy,plasma membrane,GPCR signaling,protein–protein interactions,pharmacological approaches
更新于2025-09-04 15:30:14
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Quantifying the Initial Unfolding of Bacteriorhodopsin Reveals Retinal Stabilization
摘要： The forces that stabilize membrane proteins remain elusive to precise quantification. Particularly important but poorly resolved are the forces present during a membrane protein’s initial unfolding, where the most native set of interactions are present. We developed a high-precision, atomic force microscopy assay to study the initial unfolding of bacteriorhodopsin. We discovered rapid near-equilibrium folding between the first three unfolding states that corresponded to the unfolding of 5 and 8 amino-acids respectively when using a cantilever optimized for 2-μs resolution. Interestingly, the third of these states was retinal stabilized and previously undetected despite being the most mechanically stable state in the whole unfolding pathway, supporting 150 pN for >1 min. We expect that this ability to measure the rapid and reversible dynamics in the initial unfolding of bacteriorhodopsin provides a platform for quantifying the energetics of membrane proteins under native-like conditions.
关键词： protein folding,single molecule force spectroscopy,site-specific bioconjugation,membrane proteins,atomic force microscopy
更新于2025-09-04 15:30:14
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High-throughput, non-equilibrium studies of single biomolecules using glass-made nanofluidic devices
摘要： Single-molecule detection schemes offer powerful means to overcome static and dynamic heterogeneity inherent to complex samples. However, probing biomolecular interactions and reactions with high throughput and time resolution remains challenging, often requiring surface-immobilized entities. Here, we introduce glass-made nanofluidic devices for the high-throughput detection of freely-diffusing single biomolecules by camera-based fluorescence microscopy. Nanochannels of 200 nm height and a width of several micrometers confine the movement of biomolecules. Using pressure-driven flow through an array of parallel nanochannels and by tracking the movement of fluorescently labelled DNA oligonucleotides, we observe conformational changes with high throughput. In a device geometry featuring a T-shaped junction of nanochannels, we drive steady-state non-equilibrium conditions by continuously mixing reactants and triggering chemical reactions. We use the device to probe the conformational equilibrium of a DNA hairpin as well as to continuously observe DNA synthesis in real time. Our platform offers a straightforward and robust method for studying reaction kinetics at the single-molecule level.
关键词： fluorescence microscopy,DNA,nanofluidic devices,single-molecule detection,biomolecular interactions
更新于2025-09-04 15:30:14
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Direct visualization of single-molecule membrane protein interactions in living cells
摘要： Interactions between membrane proteins are poorly understood despite their importance in cell signaling and drug development. Here, we present a co-immunoimmobilization assay (Co-II) enabling the direct observation of membrane protein interactions in single living cells that overcomes the limitations of currently prevalent proximity-based indirect methods. Using Co-II, we investigated the transient homodimerizations of epidermal growth factor receptor (EGFR) and beta-2 adrenergic receptor (β2-AR) in living cells, revealing the differential regulation of these receptors’ dimerizations by molecular conformations and microenvironment in a plasma membrane. Co-II should provide a simple, rapid, and robust platform for visualizing both weak and strong protein interactions in the plasma membrane of living cells.
关键词： co-immunoimmobilization assay,β2-AR,single-molecule visualization,living cells,membrane protein interactions,EGFR
更新于2025-09-04 15:30:14
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Wide-Range UV-to-Visible Excitation of Near-Infrared Emission and Slow Magnetic Relaxation in Ln <sup>III</sup> (4,4′-Azopyridine-1,1′-dioxide)[Co <sup>III</sup> (CN) <sub/>6</sub> ] <sup>3–</sup> Layered Frameworks
摘要： Trivalent lanthanide ions combined with two linkers, organic 4,4′-azopyridine-1,1′-dioxide (apdo), and inorganic hexacyanidocobaltate(III), gave a series of magnetoluminescent coordination polymers, [{LnIII(apdo)(H2O)4}{CoIII(CN)6}]·2H2O (Ln = Nd, 1; Tb, 2; Dy, 3; Er, 4; Tm, 5; Yb, 6). They are hybrid organic?inorganic layered frameworks composed of cyanido-bridged {Ln2(μ-NC)4Co2} squares linked by Ln?apdo?Ln bridges into a coordination network of a mixed 4- and 8-metal ring topology. Lanthanide(III) complexes, [LnIII(μ-apdo)2(H2O)4(μ-NC)2]+, of a distorted dodecahedral geometry are isolated by diamagnetic [CoIII(CN)6]3? and apdo linkers. As a result, 1?6 reveal field-induced slow relaxation of magnetization, with typical temperature-dependent relaxation of a single-ion origin for NdIII-containing 1, DyIII-containing 3, and YbIII-containing 6. The related alternate-current magnetic data were precisely analyzed, indicating the multiple magnetic relaxation pathways, including a direct process, strong quantum tunneling of magnetization, non-negligible Raman processes, and crucial two-phonon Orbach thermal relaxation. The thermal energy barriers of the Orbach process, ΔE/kB, are 15.1(9) K with τ0 = 9.8(9) × 10?6 s at Hdc = 4500 Oe, 16.1(8) K with τ0 = 9.0(9) × 10?5 s at Hdc = 1500 Oe, and 17.3(6) K with τ0 = 3.2(7) × 10?6 s at Hdc = 700 Oe, for 1, 3, and 6, respectively, proving the single-molecule magnet (SMM) behavior. Because of the presence of [Co(CN)6]3?, 1?6 show strong UV absorption, while the chromophoric apdo leads to the strong absorption in the visible range. As a result, the visible 4f/3d metal-centered emission is quenched, but the near-infrared luminescence from NdIII and YbIII is observed in 1 and 6, respectively. It is realized by Co-to-Ln metal-to-metal, and apdo-to-Ln ligand-to-metal energy transfers; thus, broad UV-to-visible excitation can be explored. Compounds 1?6 form a novel family of functional bimetallic assemblies, incorporating NIR-emissive SMMs as presented for NdCo (1) and YbCo (6) derivatives.
关键词： single-molecule magnets,near-infrared luminescence,coordination polymers,magnetoluminescent,lanthanide
更新于2025-09-04 15:30:14
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Ultra-Sensitive and Label-Free Probing of Binding Affinity Using Recognition Imaging
摘要： Reliable quantification of binding affinity is important in biotechnology and pharmacology and increasingly coupled with a demand for ultrasensitivity, nanoscale resolution, and minute sample amounts. Standard techniques are not able to meet these criteria. This study provides a new platform based on atomic force microscopy (AFM)-derived recognition imaging to determine affinity by visualizing single molecular bindings on nanosize dendrons. Using DNA hybridization as a demonstrator, an AFM sensor adorned with a cognate binding strand senses and localizes target DNAs at nanometer resolution. To overcome the limitations of speed and resolution, the AFM cantilever is sinusoidally oscillated close to resonance conditions at small amplitudes. The equilibrium dissociation constant of capturing DNA duplexes was obtained, yielding 2.4 × 10?10 M. Our label-free single-molecular biochemical analysis approach evidences the utility of recognition imaging and analysis in quantifying biomolecular interactions of just a few hundred molecules.
关键词： scanning probe microscopy,DNA hybridization,Affinity,molecular recognition,single-molecule
更新于2025-09-04 15:30:14
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Choosing the right label for single-molecule tracking in live bacteria: side-by-side comparison of photoactivatable fluorescent protein and Halo tag dyes
摘要： Visualizing and quantifying molecular motion and interactions inside living cells provides crucial insight into the mechanisms underlying cell function. This has been achieved by super-resolution localization microscopy and single-molecule tracking in conjunction with photoactivatable fluorescent proteins (PA-FPs). An alternative labelling approach relies on genetically-encoded protein tags with cell-permeable fluorescent ligands which are brighter and less prone to photobleaching than fluorescent proteins but require a laborious labelling process. Either labelling method is associated with significant advantages and disadvantages that should be taken into consideration depending on the microscopy experiment planned. Here, we describe an optimised procedure for labelling Halo-tagged proteins in live Escherichia coli cells. We provide a side-by-side comparison of Halo tag with different fluorescent ligands against the popular photoactivatable fluorescent protein PAmCherry. Using test proteins with different intracellular dynamics, we evaluated fluorescence intensity, background, photostability, and results from single-molecule localization and tracking experiments. Capitalising on the brightness and extended spectral range of fluorescent Halo ligands, we also demonstrate high-speed and dual-colour single-molecule tracking.
关键词： Halo tag,single-molecule tracking,photoactivatable fluorescent protein,Escherichia coli,fluorophores,super-resolution microscopy,DNA-binding proteins
更新于2025-09-04 15:30:14
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Single-Molecule Kinetic Fingerprinting for the Ultrasensitive Detection of Small Molecules with Aptasensors
摘要： Aptamers have emerged as promising molecular tools for small-molecule analyte sensing. However, the performance of such aptasensors is generally limited by leakage since it has been difficult to completely suppress signal in the absence of analyte, resulting in a compromise between sensitivity and specificity. Here, we describe a methodology for the ultrasensitive detection of analytes combining aptasensors with single-molecule kinetic fingerprinting. A short, fluorescently labeled DNA probe is utilized to detect the structural changes upon ligand binding to the designed hairpin-shaped aptasensor probe. The Poisson statistics of binding and dissociation events of the DNA probe to single surface-immobilized aptasensor molecules is monitored by total internal reflection fluorescence microscopy, permitting the high-accuracy discrimination of the ligand bound and ligand-free states, resulting in zero background. The programmable dynamics of the hairpin enables fine-tuning of the hybridization kinetics of the fluorescent probe, rendering the acquisition time sufficiently flexible to optimize discrimination. Remarkable detection limits are achieved for a diverse set of analytes when spiked into chicken meat extract: the nucleotide adenosine (0.3 pM), the insecticide acetamiprid (0.35 pM), and the dioxin-like toxin PCB-77 (0.72 pM), which is superior to recently reported aptasensors. Our generalizable method significantly improves the performance of aptasensors, with the potential to extend to other molecular biomarkers.
关键词： Aptamers,Aptasensors,Single-molecule kinetic fingerprinting,Ultrasensitive detection,Small molecules
更新于2025-09-04 15:30:14