Characterizing rhodopsin signaling by EPR spectroscopy: from structure to dynamics

Eps, Ned Van; Lydia, Ng; Morizumi, Takefumi; Ernst, Oliver P.

doi:10.1039/c5pp00191a

Cited by 18 publications

(10 citation statements)

References 90 publications

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“…It is unclear what the precise structural differences are that define the various active states in rhodopsin implied by the data presented in the current study. The ability of rhodopsin to adopt multiple active states has been demonstrated by double electron-electron resonance spectroscopy on rhodopsin reconstituted into nanodiscs 50 . Electrophysiological measurements in photoreceptor cells expressing G90D rhodopsin have demonstrated the possibility that G90D rhodopsin adopts an active conformation different from the lightactivated MII state 47 .…”

Section: Conformational State Of Constitutively Active Forms Of Rhodomentioning

confidence: 99%

Differentiating between Inactive and Active States of Rhodopsin by Atomic Force Microscopy in Native Membranes

et al. 2019

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Membrane proteins, including G protein-coupled receptors (GPCRs), present a challenge in studying their structural properties under physiological conditions. Moreover, to better understand the activity of proteins requires examination of single molecule behaviors rather than ensemble averaged behaviors. Force-distance curve-based AFM (FD-AFM) was utilized to directly probe and localize the conformational states of a GPCR within the membrane at nanoscale resolution based on the mechanical properties of the receptor. FD-AFM was applied to rhodopsin, the light receptor and a prototypical GPCR, embedded in native rod outer segment disc membranes from photoreceptor cells of the retina in mice. Both FD-AFM and computational studies on coarsegrained models of rhodopsin revealed that the active state of the receptor has a higher Young's modulus compared to the inactive state of the receptor. Thus, the inactive and active states of rhodopsin could be differentiated based on the stiffness of the receptor. Differentiating the states based on the Young's modulus allowed for the mapping of the different states within the membrane. Quantifying the active states present in the membrane containing the constitutively active G90D rhodopsin mutant or apoprotein opsin revealed that most receptors adopt an active state. Traditionally, constitutive activity of GPCRs has been described in terms of two-state models *

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Section: Conformational State Of Constitutively Active Forms Of Rhodomentioning

confidence: 99%

Differentiating between Inactive and Active States of Rhodopsin by Atomic Force Microscopy in Native Membranes

et al. 2019

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“…Pulsed EPR (DEER) was first applied to rhodopsin [ 248 , 249 , 250 ] and has revealed the pattern of helix movements that accompany receptor activation, which include a 5 Å outward movement of TM6 and smaller changes for TM1 and TM7, while TM3 does not move [ 249 ]. In contrast to most other GPCRs, rhodopsin displays a stronger allosteric connection between the ligand binding pocket and its intracellular interaction interface, and follows a stricter sequential order of activation events [ 36 , 251 ], which likely originates from its specialized function.…”

Section: A Receptor’s Perspectivementioning

confidence: 99%

Capturing Peptide–GPCR Interactions and Their Dynamics

Kaiser

Coin

2020

Molecules

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Many biological functions of peptides are mediated through G protein-coupled receptors (GPCRs). Upon ligand binding, GPCRs undergo conformational changes that facilitate the binding and activation of multiple effectors. GPCRs regulate nearly all physiological processes and are a favorite pharmacological target. In particular, drugs are sought after that elicit the recruitment of selected effectors only (biased ligands). Understanding how ligands bind to GPCRs and which conformational changes they induce is a fundamental step toward the development of more efficient and specific drugs. Moreover, it is emerging that the dynamic of the ligand–receptor interaction contributes to the specificity of both ligand recognition and effector recruitment, an aspect that is missing in structural snapshots from crystallography. We describe here biochemical and biophysical techniques to address ligand–receptor interactions in their structural and dynamic aspects, which include mutagenesis, crosslinking, spectroscopic techniques, and mass-spectrometry profiling. With a main focus on peptide receptors, we present methods to unveil the ligand–receptor contact interface and methods that address conformational changes both in the ligand and the GPCR. The presented studies highlight a wide structural heterogeneity among peptide receptors, reveal distinct structural changes occurring during ligand binding and a surprisingly high dynamics of the ligand–GPCR complexes.

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“…Advancements in pulsed-EPR techniques, such as double electron-electron resonance (DEER), provide an opportunity to refine GPCR structures obtained by X-ray crystallography or, more recently, cryo electron microscopy 18 , while simultaneously identifying distributions of conformations constituting the ensemble 19-20 . Moreover, this can be repeated under various conditions (i.e., pH, ligand, detergent, liposomes, nanodiscs etc.) 21 . At the same time, EPR measurements provide a measure of dynamics of specific states, particularly on nanosecond and microsecond timescales.…”

Section: Introductionmentioning

confidence: 99%

Ligand Modulation of the Conformational Dynamics of the A_2AAdenosine Receptor Revealed by Single-Molecule Fluorescence

Fernandes

Neale

Gomes

et al. 2020

Preprint

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G protein-coupled receptors (GPCRs) are the largest class of transmembrane proteins, making them an important target for therapeutics. Activation of these receptors is modulated by orthosteric ligands, which stabilize one or several states within a complex conformational ensemble. The intra-and inter-state dynamics, however, is not well documented. Here, we used single-molecule fluorescence to measure ligand-modulated conformational dynamics of the adenosine A2A Receptor (A2AR) on nanosecond to millisecond timescales. Experiments were performed on detergent-purified A2R in either the ligand-free (apo) state, or when bound to an inverse, partial or full agonist ligand. Single-molecule Förster resonance energy transfer (smFRET) was performed on detergent-solubilized A2AR to resolve active and inactive states via the separation between transmembrane (TM) helices 4 and 6. The ligand-dependent changes of the smFRET distributions are consistent with conformational selection and with inter-state exchange lifetimes ≥ 3 ms. Local conformational dynamics around residue 229 on TM6 was measured using Fluorescence Correlation Spectroscopy (FCS), which captures dynamic quenching due to photoinduced electron transfer (PET) between a covalently-attached dye and proximal aromatic residues. Global analysis of PET-FCS data revealed fast (150-350 ns), intermediate (50-60 μs) and slow (200-300 μs) conformational dynamics in A2AR, with lifetimes and amplitudes modulated by ligands and a G-protein mimetic (mini-Gs). Most notably, the agonist binding and the coupling to mini-Gs accelerates and increases the relative contribution of the sub-microsecond phase. Molecular dynamics simulations identified three tyrosine residues (Y112, Y288, and Y290) as being responsible for the dynamic quenching observed by PET-FCS and revealed associated helical motions around residue 229 on TM6. This study provides a quantitative description of conformational dynamics in A2AR and supports the idea that ligands bias not only GPCR conformations but also the dynamics within and between distinct conformational states of the receptor.

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Characterizing rhodopsin signaling by EPR spectroscopy: from structure to dynamics

Cited by 18 publications

References 90 publications

Differentiating between Inactive and Active States of Rhodopsin by Atomic Force Microscopy in Native Membranes

Differentiating between Inactive and Active States of Rhodopsin by Atomic Force Microscopy in Native Membranes

Capturing Peptide–GPCR Interactions and Their Dynamics

Ligand Modulation of the Conformational Dynamics of the A_2AAdenosine Receptor Revealed by Single-Molecule Fluorescence

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Characterizing rhodopsin signaling by EPR spectroscopy: from structure to dynamics

Cited by 18 publications

References 90 publications

Differentiating between Inactive and Active States of Rhodopsin by Atomic Force Microscopy in Native Membranes

Differentiating between Inactive and Active States of Rhodopsin by Atomic Force Microscopy in Native Membranes

Capturing Peptide–GPCR Interactions and Their Dynamics

Ligand Modulation of the Conformational Dynamics of the A2AAdenosine Receptor Revealed by Single-Molecule Fluorescence

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Product

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Ligand Modulation of the Conformational Dynamics of the A_2AAdenosine Receptor Revealed by Single-Molecule Fluorescence