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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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Diarylethene moiety as an enthalpy-entropy switch: photoisomerizable stapled peptides for modulating p53/MDM2 interaction
摘要： Analogs of the known inhibitor (peptide pDI) of the p53/MDM2 protein–protein interaction are reported, which are stapled by linkers bearing a photoisomerizable diarylethene moiety. The corresponding photoisomers possess significantly different affinities to the p53-interacting domain of the human MDM2. Apparent dissociation constants are in the picomolar-to-low nanomolar range for those isomers with diarylethene in the 'open' configuration, but up to eight times larger for the corresponding 'closed' isomers. Spectroscopic, structural, and computational studies showed that the stapling linkers of the peptides contribute to their binding. Calorimetry revealed that the binding of the 'closed' isomers is mostly enthalpy-driven, whereas the 'open' photoforms bind to the protein stronger due to their increased binding entropy. The results suggest that conformational dynamics of the protein-peptide complexes may explain the differences in the thermodynamic profiles of the binding.
关键词： Photoisomerizable stapled peptides,Thermodynamic profiles,p53/MDM2 interaction,Diarylethene,Conformational dynamics
更新于2025-09-23 15:21:01
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[Methods in Enzymology] || Fluorescence Depolarization Kinetics to Study the Conformational Preference, Structural Plasticity, Binding, and Assembly of Intrinsically Disordered Proteins
摘要： Fluorescence depolarization kinetics measured by the time-resolved fluorescence anisotropy decay serves as a sensitive and powerful methodology to study the conformational dynamics of macromolecules. This methodology allows us to delineate the different modes of biomolecular motional dynamics including the local, segmental, and global rotational dynamics on the timescale ranging from picoseconds to nanoseconds. In this chapter, we describe the principles and applications of this methodology to obtain unique molecular insights into the intrinsically disordered proteins (IDPs). Fluorescence depolarization kinetics, when performed in a site-specific manner, can offer a reliable tool to monitor the intrinsic backbone torsional dynamics of expanded IDPs and is capable of discerning the conformational preference of IDPs. Additionally, the time-resolved fluorescence anisotropy measurements allow us to investigate the mechanism of binding and assembly of a wide range of IDPs that are involved in crucial function and disease.
关键词： Fluorescence depolarization kinetics,conformational dynamics,time-resolved fluorescence anisotropy decay,biomolecular interactions,intrinsically disordered proteins
更新于2025-09-23 15:21:01
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Revealing Conformational Transitions in G-Protein Coupled Receptor Rhodopsin upon Phosphorylation
摘要： GPCRs have evolved as highly specialized cellular machinery that can dictate biological outcomes in response to diverse stimuli. Specifically, they induce multiple pathway responses upon structural perturbations induced at local protein sites. GPCRs utilize concurrent strategy involving a central transmembrane topology and biochemical modifications for precise functional implementation. However, the specific role of latter is not decomposed due to the lack of precise probing techniques that can characterize receptor dynamics upon biochemical modifications. Phosphorylation is known to be one of the critical biochemical modifications in GPCRs that aids in receptor desensitization via arrestin binding. Here, we carried out all-atom molecular dynamics (MD) simulations of rhodopsin in membrane environment to study its conformational dynamics induced upon phosphorylation. Interestingly, our comparative analysis of non-phosphorylated and phosphorylated rhodopsin structure demonstrated enhanced receptor stability upon phosphorylation at the C-terminal region that leads to the opening of the extracellular part of the transmembrane helices. In addition, monitoring of the distinct number of phosphorylation states showed that less number of phosphorylated residues does not bring about appropriate conformational changes in the extracellular region. Since phosphorylation results in receptor desensitization and recycling of ligand, our findings provide significant insights into the conformational dynamics of the mechanism of ligand exit from the receptor.
关键词： conformational dynamics,molecular dynamics simulations,GPCRs,phosphorylation,rhodopsin
更新于2025-09-12 10:27:22