Investigation of Rhodopsin Dynamics in Its Signaling State by Solid-State Deuterium NMR Spectroscopy

Struts, Andrey V.; Chawla, Udeep; Perera, Suchithranga M.D.C.; Brown, Michael F.

doi:10.1007/978-1-4939-2330-4_10

Cited by 6 publications

(9 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…29 The sample was photobleached with green LED light of 515 nm, which activates rhodopsin to form metarhodopsin-II. 41,51 Including NH 2 OH ensures complete photobleaching of the dark-state rhodopsin and is confirmed by the nearly zero absorbance at ca. 500 nm (Figure S1; see SI for further details on preparation and characterization of powdered RDM samples).…”

Section: ■ Materials and Methodsmentioning

confidence: 84%

“…Retinal disk membranes (RDM) containing rhodopsin were isolated from bovine retinas as described. 41 The RDM pellet was resuspended in 15 mM sodium phosphate buffer, pH 6.9, and characterized using UV-visible spectroscopy 51 to determine the level of purity ( A 280 / A 500 absorption ratio was typically 2.4). The rhodopsin concentration was estimated from the absorbance of the sample at 500 nm.…”

Section: Methodsmentioning

confidence: 99%

“…However, thus far little information is available regarding how the internal dynamics of the protein change during GPCR activation. 41 Application of the powdered GPCR preparation method together with use of neutron scattering techniques allows for the study of changes in the protein dynamics upon rhodopsin activation. Current findings suggest the intrinsic dynamics of the protein are unlocked by the light-induced isomerization of the 11- cis retinal cofactor needed for interaction of the GPCR with its cognate G-protein (transducin).…”

mentioning

confidence: 99%

See 2 more Smart Citations

Powdered G-Protein-Coupled Receptors

Perera

Chawla

Brown

2016

J. Phys. Chem. Lett.

Self Cite

View full text Add to dashboard Cite

Preparation and storage of functional membrane proteins such as G-protein-coupled receptors (GPCRs) are crucial to the processes of drug delivery and discovery. Here we describe a method of preparing powdered GPCRs using rhodopsin as the prototype. We purified rhodopsin in CHAPS detergent with low detergent to protein ratio so the bulk of the sample represented protein (ca. 72% w/w). Our new method for generating powders of membrane proteins followed by rehydration paves the way for conducting functional and biophysical experiments. As an illustrative application powdered rhodopsin was prepared with and without the cofactor 11-cis retinal to enable partial rehydration of the protein with D2O in a controlled manner. Quasielastic neutron scattering studies using both spatial motion and energy landscape models form the basis for crucial insights into structural fluctuations and thermodynamics of GPCR activation.

show abstract

Section: ■ Materials and Methodsmentioning

confidence: 84%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Powdered G-Protein-Coupled Receptors

Perera

Chawla

Brown

2016

J. Phys. Chem. Lett.

Self Cite

View full text Add to dashboard Cite

show abstract

“…This lack of dynamical information and structural perturbation makes an interpretation of functional data directly from these structures a challenge and often alternate/complementary methods are employed. For example, along with traditionally employed biophysical methods such as site-directed spin labeling and disulfide cross-linking (Farrens et al, 1996), NMR (Soubias & Gawrisch, 2012; Struts, Chawla, Perera, & Brown, 2015), BRET/FRET (Lohse, Nuber, & Hoffmann, 2012), double electron–electron resonance (Altenbach, Kusnetzow, Ernst, Hofmann, & Hubbell, 2008), and fluorescence (Fay & Farrens, 2015) spectroscopies, as well as molecular dynamics (MD) simulations have become pivotal in characterizing these systems (Grossfield, 2011; Latorraca et al, 2017; Marino, Shang, & Filizola, 2017). …”

Section: Introductionmentioning

confidence: 99%

Molecular Dynamics Methodologies for Probing Cannabinoid Ligand/Receptor Interaction

Lynch

Hurst

Shore

et al. 2017

Methods in Enzymology

View full text Add to dashboard Cite

The cannabinoid type 1 and 2 G-protein-coupled receptors are currently important pharmacological targets with significant drug discovery potential. These receptors have been shown to display functional selectivity or biased agonism, a property currently thought to have substantial therapeutic potential. Although recent advances in crystallization techniques have provided a wealth of structural information about this important class of membrane-embedded proteins, these structures lack dynamical information. In order to fully understand the interplay of structure and function for this important class of proteins, complementary techniques that address the dynamical aspects of their function are required such as NMR as well as a variety of other spectroscopies. Complimentary to these experimental approaches is molecular dynamics, which has been effectively used to help unravel, at the atomic level, the dynamics of ligand binding and activation of these membrane-bound receptors. Here, we discuss and present several representative examples of the application of molecular dynamics simulations to the understanding of the signatures of ligand-binding and -biased signaling at the cannabinoid type 1 and 2 receptors.

show abstract

“…It can be used to characterize membrane-bound structures (20,21), and it can yield detailed information on the conformation and dynamics of membrane-bound proteins and peptides (22,23,24) and their artificial mimics (25). It has recently been used to investigate both the structure and dynamics of the photoswitchable vision protein rhodopsin (26), and provides evidence that the solution-phase conformational preference of the Aib-rich peptaibol alamethicin is preserved when embedded in phospholipid bilayers (27). Magic angle spinning (MAS) is the most commonly used ss-NMR method (28), and in a lipid bilayer, MAS spin rates of ~10 kHz generate well resolved 1 H ss-NMR signals within an experimental time scale of 1 h (29).…”

mentioning

confidence: 99%

Conformational photoswitching of a synthetic peptide foldamer bound within a phospholipid bilayer

Poli

Zawodny

Quinonero

et al. 2016

Science

152

137

View full text Add to dashboard Cite

The dynamic properties of foldamers, synthetic molecules that mimic folded biomolecules, have mainly been explored in free solution. We report on the design, synthesis, and conformational behavior of photoresponsive foldamers bound in a phospholipid bilayer akin to a biological membrane phase. These molecules contain a chromophore, which can be switched between two configurations by different wavelengths of light, attached to a helical synthetic peptide that both promotes membrane insertion and communicates conformational change along its length. Light-induced structural changes in the chromophore are translated into global conformational changes, which are detected by monitoring the solid-state (19)F nuclear magnetic resonance signals of a remote fluorine-containing residue located 1 to 2 nanometers away. The behavior of the foldamers in the membrane phase is similar to that of analogous compounds in organic solvents.

show abstract

Investigation of Rhodopsin Dynamics in Its Signaling State by Solid-State Deuterium NMR Spectroscopy

Cited by 6 publications

References 35 publications

Powdered G-Protein-Coupled Receptors

Powdered G-Protein-Coupled Receptors

Molecular Dynamics Methodologies for Probing Cannabinoid Ligand/Receptor Interaction

Conformational photoswitching of a synthetic peptide foldamer bound within a phospholipid bilayer

Contact Info

Product

Resources

About