Spin-label saturation-transfer electron spin resonance detection of transient association of rhodopsin in reconstituted membranes

Kusumi, Akihiro; Hyde, James S.

doi:10.1021/bi00266a039

Cited by 98 publications

(89 citation statements)

References 56 publications

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“…ESR studies of the type reported here with spin-labelled lipids in combination with saturation transfer experiments of spin-labelled bovine rhodopsin showed that varying the hydrophobic thickness of the bilayer could induce extensive aggregation or an oligomeric arrangement of the receptor [63,67]. More recently, FRET studies with Alexa fluorophores attached to bovine rhodopsin also showed a dependence of receptor oligomerisation/ aggregation on lipid chain length [68].…”

Section: Effect Of Increasing the Thickness Of The Lipid Bilayermentioning

confidence: 53%

Interaction of lipids with the neurotensin receptor 1

Bolivar

Muñoz–García

Castro‐Dopico

et al. 2016

Biochimica et Biophysica Acta (BBA) - Biomembranes

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Information about lipid-protein interactions for G protein-coupled receptors (GPCRs) is scarce. Here, we use electron spin resonance (ESR) and spin-labelled lipids to study lipid interactions with the rat neurotensin receptor 1 (NTS1). A fusion protein containing rat NTS1 fully able to bind its ligand neurotensin was reconstituted into phosphatidylcholine (PC) bilayers at specific lipid:protein molar ratios. The fraction of motionally restricted lipids in the range of 40:1 to 80:1 lipids per receptor suggested an oligomeric state of the protein, and the result was unaffected by increasing the hydrophobic thickness of the lipid bilayer from C-18 to C-20 or C-22 chain length PC membranes. Comparison of the ESR spectra of different spin-labelled lipids allowed direct measurement of lipid binding constants relative to PC (Kr), with spin-labelled phosphatidylethanolamine (PESL), phosphatidylserine (PSSL), stearic acid (SASL), and a spin labelled cholesterol analogue (CSL) Kr values of 1.05±0.05, 1.92±0.08, 5.20±0.51 and 0.91±0.19, respectively. The results contrast with those from rhodopsin, the only other GPCR studied this way, which has no selectivity for the lipids analysed here. Molecular dynamics simulations of NTS1 in bilayers are in agreement with the ESR data, and point to sites in the receptor where PS could interact with higher affinity. Lipid selectivity could be necessary for regulation of ligand binding, oligomerisation and/or G protein activation processes. Our results provide insight into the potential modulatory mechanisms that lipids can exert on GPCRs.

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Section: Effect Of Increasing the Thickness Of The Lipid Bilayermentioning

confidence: 53%

Interaction of lipids with the neurotensin receptor 1

Bolivar

Muñoz–García

Castro‐Dopico

et al. 2016

Biochimica et Biophysica Acta (BBA) - Biomembranes

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“…Powder-type 2 H NMR spectra at temperatures of −30, −60, and −150 °C are similar and exhibit discontinuities at ∓19.5 kHz and ±39.0 kHz, due to the θ = 90° and θ = 0° orientations of the largest principal value of the electric field gradient (EFG) tensor relative to the main magnetic field 24 . Below the order-disorder phase transition temperature of POPC (T M = −4 °C) the 2 H NMR spectra correspond to the lipid gel state, where rotational and translational diffusion of both the protein 35 and lipids are diminished on the 2 H NMR time scale (< 10 μs for rotating methyls). Weak signals from the residual 1 HO 2 H of the solvent are subtracted at T = −30 and −60 °C (not shown).…”

Section: Powder-type 2 H Nmr Spectra Of Retinal Bound To Rhodopsinmentioning

confidence: 99%

“…In gel-state bilayers, membrane proteins do not undergo significant rotational diffusion 35 on the 2 H NMR time scale (≈10 μL). The quadrupolar frequencies of the two spectral branches read 24 (A1)…”

Section: Spectral Lineshape Analysis For Static Axial Distributionmentioning

confidence: 99%

Structural Analysis and Dynamics of Retinal Chromophore in Dark and Meta I States of Rhodopsin from 2H NMR of Aligned Membranes

Struts

Salgado

Tanaka

et al. 2007

Journal of Molecular Biology

134

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SummaryRhodopsin is a prototype for G protein-coupled receptors (GPCRs) that are implicated in many biological responses in humans. A site-directed 2 H NMR approach was used for structural analysis of retinal within its binding cavity in the dark and pre-activated meta I states. Retinal was labeled with 2 H at the C5, C9, or C13 methyl groups by total synthesis, and was used to regenerate the opsin apoprotein. Solid-state 2 H NMR spectra were acquired for aligned membranes in the lowtemperature lipid gel phase versus the tilt angle to the magnetic field. Data reduction assumed a static uniaxial distribution, and gave the retinylidene methyl bond orientations together with the alignment disorder (mosaic spread). The 2 H NMR structure of 11-cis-retinal in the dark state revealed torsional twisting of the polyene chain and the β-ionone ring. The distorted retinylidene ligand undergoes restricted motion, as evinced by order parameters of ≈ 0.9 for the rapidly spinning C-C 2 H 3 groups, with off-axial fluctuations of ≈ 15°. Retinal is accommodated within the rhodopsin binding pocket with a negative pre-twist about the C11=C12 double bond, which explains its rapid photochemistry and indicates the trajectory of the 11-cis to trans isomerization.For the cryotrapped meta I state, the 2 H NMR structure showed a reduction of the polyene strain, whereas the β-ionone ring maintained its torsional twisting. Strain energy and dynamics of retinal are interpreted with regard to substituent control of receptor activation. Steric hindrance between trans retinal and Trp 265 can trigger formation of the subsequent activated meta II state. Our results are pertinent to quantum and classical molecular mechanics simulations, and show how 2 H NMR can be applied to ligands bound to GPCRs in relation to their characteristic mechanisms of action.

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“…The low signal-to-noise ratio of the decay curves prevented us from carryingouta detailedanalysistodetectthesmallchangesin therotational diffusion ratethat may beinducedby transient associationof BR (Kusumi & Hyde, 1982). Saturation transfer ESRspectroscopy was not performed because the spin labels could not be strongly immobilized on the protein (nor has any report been published on saturation transfer spectroscopy of spin-labeled BR).…”

Section: Self-assmiationstates Of Br In Reconstituted Membranesmentioning

confidence: 99%

Molecular Organization and Dynamics in Bacteriorhodopsin-Rich Reconstituted Membranes: Discrimination of Lipid Environments by the Oxygen Transport Parameter Using a Pulse ESR Spin-Labeling Technique

Ashikawa

Yin²,

Subczynski³

et al. 1994

Biochemistry

Self Cite

136

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Molecular organization and dynamics in protein-rich membranes have been studied by investigating transport (diffusion-concentration product) of molecular oxygen at various locations in reconstituted membranes of bacteriorhodopsin (BR) and L-alpha-dimyristoylphosphatidylcholine. Oxygen transport was evaluated by monitoring the bimolecular collision of molecular oxygen with four types of nitroxide lipid spin labels placed at various locations in the membrane. The collision rate was estimated from the spin-lattice relaxation times (T1's) measured at various oxygen partial pressures by analyzing the short-pulse saturation recovery ESR signals. CD spectra and decay of polarized flash-induced photodichroism of bacteriorhodopsin indicated that BR molecules are monomers in reconstituted membranes with a lipid/BR molar ratio of 80 (80-rec) and are 25% monomers and 75% trimers plus oligomers of trimers when the lipid/BR ratio is 40 (40-rec). In the 80-rec, the lipid environment is homogeneous on a microsecond scale (T1), probably because the exchange rate of lipids between the bulk and the boundary regions is greater than the T1 relaxation rate (approximately 10(6) s-1). The oxygen collision rate in the hydrophobic region of the 80-rec membrane is smaller by a factor of 1.6 than in that of the lipid membrane without BR, and the effect of BR in decreasing the collision rate is independent of the "depth" in the hydrophobic region. In the 40-rec, two collision rates were observed, one of which is close to those for purple membrane (or the gel-phase membrane), while the other is about the same as was measured in the 80-rec.(ABSTRACT TRUNCATED AT 250 WORDS)

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Spin-label saturation-transfer electron spin resonance detection of transient association of rhodopsin in reconstituted membranes

Cited by 98 publications

References 56 publications

Interaction of lipids with the neurotensin receptor 1

Interaction of lipids with the neurotensin receptor 1

Structural Analysis and Dynamics of Retinal Chromophore in Dark and Meta I States of Rhodopsin from 2H NMR of Aligned Membranes

Molecular Organization and Dynamics in Bacteriorhodopsin-Rich Reconstituted Membranes: Discrimination of Lipid Environments by the Oxygen Transport Parameter Using a Pulse ESR Spin-Labeling Technique

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