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Fluorophore Labeling, Nanodisc Reconstitution and Single-molecule Observation of a G Protein-coupled Receptor
摘要： Activation of G protein-coupled receptors (GPCRs) by agonist ligands is mediated by a transition from an inactive to active receptor conformation. We describe a novel single-molecule assay that monitors activation-linked conformational transitions in individual GPCR molecules in real-time. The receptor is site-specifically labeled with a Cy3 fluorescence probe at the end of trans-membrane helix 6 and reconstituted in phospholipid nanodiscs tethered to a microscope slide. Individual receptor molecules are then monitored over time by single-molecule total internal reflection fluorescence microscopy, revealing spontaneous transitions between inactive and active-like conformations. The assay provides information on the equilibrium distribution of inactive and active receptor conformations and the rate constants for conformational exchange. The experiments can be performed in the absence of ligands, revealing the spontaneous conformational transitions responsible for basal signaling activity, or in the presence of agonist or inverse agonist ligands, revealing how the ligands alter the dynamics of the receptor to either stimulate or repress signaling activity. The resulting mechanistic information is useful for the design of improved GPCR-targeting drugs. The single-molecule assay is described in the context of the β2 adrenergic receptor, but can be extended to a variety of GPCRs.
关键词： Phospholipid nanodiscs,G-protein coupled receptors,Conformational dynamics,β2 adrenergic receptor,Single-molecule fluorescence
更新于2025-11-21 11:24:58
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Ultrathin Y <sub/>2</sub> O <sub/>3</sub> :Eu <sup>3+</sup> nanodiscs: spectroscopic investigations and evidence for reduced concentration quenching
摘要： Here, we report the synthesis and spectral properties of ultrathin nanodiscs (NDs) of Y2O3:Eu3+. It was found that the NDs of Y2O3:Eu3+ with a thickness of about 1 nm can be fabricated in a reproducible, facile and self-assembling process, which does not depend on the Eu3+ concentration. The thickness and morphology of these NDs were determined with small angle x-ray scattering and transmission electron microscopy. We found that the crystal field in these nanoparticles deviates from both the cubic and monoclinic characteristics, albeit the shape of the 5D0 → 7FJ (J = 0, 1, 2) transitions shows some similarity with the transitions in the monoclinic material. The Raman spectra of the non-annealed NDs manifest various vibration modes of the oleic acid molecules, which are used to stabilise the NDs. The annealed NDs show two very weak Raman lines, which may be assigned to vibrational modes of Y2O3 NDs. The concentration quenching of the Eu3+ luminescence of the NDs before annealing is largely suppressed and might be explained in terms of a reduction of the phonon density of states.
关键词： nanodiscs,small angle x-ray scattering,europium-doped yttrium oxide,Raman spectroscopy,concentration quenching,transmission electron microscopy,luminescence
更新于2025-09-23 15:21:21
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Nanodisc decorated W-WOx suspended nanowire: A highly sensitive and selective H2S sensor
摘要： In this work we report room temperature synthesis of plasma oxidized, suspended tungsten-tungsten oxide (W-WOx) core-shell nanowire for sensing ppb level H2S. The electric field modulation at the W-WOx interface of the core-shell nanowire strongly influences the sensing performance and brings down the operating temperature all the way down to 50°C, compared to completely oxidized (WOx) nanowire. The optimum interface ratio (W/WOx) of the nanowire shows response of 90.4% (1 ppm) with 6 months of response stability and excellent selectivity. The limit of detection of 10 ppb with response and recovery time of 4 s and 46 s respectively is achieved. To enhance the response further, we utilize nanostructuring on top of nanowire, using nanodiscs of 20 nm, 50 nm and 100 nm diameter and 10 nm height. The nanowire with nanodiscs of 20 nm diameter shows the high repeatable response of 12529% (1 ppm) at 150°C and fast response and recovery times of 12 s and 19 s with detection limit of 0.5 ppb. As we switch from unpatterned to patterned nanowire the observed change in H2S sensing characteristics, indicates that the core-shell nanowire behaviour makes a transition from p-type to n-type. Extensive material characterization is done using UV-Vis spectroscopy, XPS and TEM.
关键词： plasma oxidation,selectivity,Core-shell nanowire,nanodiscs,suspended
更新于2025-09-09 09:28:46