Investigation of rhodopsin dynamics in its signaling state by solid-state deuterium NMR spectroscopy - PubMed (original) (raw)

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Investigation of rhodopsin dynamics in its signaling state by solid-state deuterium NMR spectroscopy

Andrey V Struts et al. Methods Mol Biol. 2015.

Abstract

Site-directed deuterium NMR spectroscopy is a valuable tool to study the structural dynamics of biomolecules in cases where solution NMR is inapplicable. Solid-state (2)H NMR spectral studies of aligned membrane samples of rhodopsin with selectively labeled retinal provide information on structural changes of the chromophore in different protein states. Moreover (2)H NMR relaxation time measurements allow one to study the dynamics of the ligand during the transition from the inactive to the active state. Here we describe the methodological aspects of solid-state (2)H NMR spectroscopy for functional studies of rhodopsin, with an emphasis on the dynamics of the retinal cofactor. We provide complete protocols for the preparation of NMR samples of rhodopsin with 11-cis-retinal selectively deuterated at the methyl groups in aligned membranes. In addition we review optimized conditions for trapping the rhodopsin photointermediates; and we address the challenging problem of trapping the signaling state of rhodopsin in aligned membrane films.

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Figures

Fig. 1

Fig. 1

Representative UV-visible absorption spectra of rhodopsin (a) in ROS disk membranes solubilized in 3% Ammonyx LO, pH 6.8, (Section 2.1), and (b) after column purification in 100 mM DTAB detergent buffer, pH 6.8, in presence of 10 mM hydroxylamine. The 11-cis retinal absorption in the dark-state rhodopsin is shifted to 500 nm due to the protonated Schiff base covalently bound to the protein, and its interaction with surrounding amino acids in the binding pocket. After bleaching in the presence of hydroxylamine, the absorption with maximum around 360 nm is due to free hydrolyzed retinal oxime. The _A_280/_A_500 absorption ratio characterizes the spectral purity of rhodopsin (see Note 3).

Fig. 2

Fig. 2

Solid-state deuterium NMR spectroscopy of selectively 2H-labeled retinal bound to rhodopsin in randomly oriented membranes. (a) Structure of the Meta II state of rhodopsin (protein databank accession code 4A4M) compared to dark-state rhodopsin (code 1U19) showing activating movement of helices. (b) Numbering scheme of retinal ligand indicating methyl groups studied. (c) Solid-state 2H NMR spectra of deuterated retinal specifically labeled at C5-, C9-, or C13-Me positions bound to rhodopsin in unoriented membranes (10). The spectra are calculated by convolution of the frequency distribution for the randomly oriented quadrupolar coupling tensor and intrinsic line broadening Δν. The spectral fitting indicates that the average quadrupolar coupling constant 〈_χQ_〉 is reduced by a factor of three due to rapid methyl group spinning, and by another 10 % due to off-axial fluctuations with respect to the average orientation (see text). The line broadening Δν is in the range of 3.2–5.0 kHz. Figure adapted from ref. (10).

Fig. 3

Fig. 3

Examples of phosphorus-31 (a,b) and deuterium NMR (c) spectra of aligned membranes containing rhodopsin with 2H-labeled retinal: (a) solid-state 31P NMR spectrum of rhodopsin-POPC (1:50 protein/lipid ratio) recombinant membranes with a largely complete alignment; (b) solid-state 31P NMR spectrum of recombinant membranes (3:1 POPC/DOPE molar ratio) with rhodopsin (1:75 protein/lipid ratio) in 5 mM 2H-depleted MES buffer (pH 5.5 at 4 °C). One can see a noticeable peak at about −20 ppm in the right-hand spectrum, corresponding to the θ = 90 ° contribution of unoriented bilayers. The experimental spectrum (blue) in Fig. 1b is fitted to the theoretical spectrum (red solid line), approximated as a superposition of a Lorenzian (corresponding to aligned fraction, indicated by black solid line) and a powder-type spectrum (dashed line). The fitting parameters are the chemical shift anisotropy (Δσ), line broadening (Δν), and intensities of the aligned and powder-type spectra. The calculated unoriented fraction is 14%. The 31P NMR spectra are measured with a Bruker AMX-500 spectrometer (magnetic field 11.7 T) at room temperature using a locally constructed (“home built”) 31P probe with a transverse solenoid coil (8-mm diameter, 10-mm length). Spectral resolution is high enough to resolve a contribution to the spectrum from unoriented bilayers even without proton decoupling. (c) Experimental and simulated 2H NMR spectra of 11-_cis_-retinal with selectively 2H-labeled C5-, C9-, or C13-Me groups bound to rhodopsin in aligned membranes. The rhodopsin/POPC molar ratio is 1:50. Experimental 2H NMR spectra are measured on a Bruker AMX-500 spectrometer (see Note 12) at −150 °C with the average bilayer normal inclined at θ = 0 ° to the magnetic field.

Fig. 4

Fig. 4

UV-visible absorption spectra of aligned membrane films containing rhodopsin (on glass slides) in the dark (dashed lines), and trapped Meta I (a) and Meta II (b) states (both states are indicated by solid lines). The Meta I state was obtained by illuminating rhodopsin-POPC recombinant membranes (1:50 protein/lipid ratio) with actinic light at 2 °C, pH 7, and 43% relative humidity. The Meta II state was trapped by bleaching recombinant membranes (3:1 POPC/DOPE molar ratio) with rhodopsin (1:75 protein/lipid ratio) in 5 mM 2H-depleted MES buffer at room temperature, pH 5.0, and 100% relative humidity. The average shape of the DOPE lipid molecule, shown on the right (bottom) corresponds to a negative spontaneous (intrinsic) monolayer curvature, which favors the Meta II state. By contrast POPC, shown on the right (top), favors flat bilayers thus shifting the metarhodopsin equilibrium back to Meta I in accord with a flexible surface model (35).

Fig. 5

Fig. 5

Inversion-recovery solid-state 2H NMR spectra (a) for methyl groups of 11-_cis_-retinal 2H-labeled at the C9- or C13-Me positions bound to rhodopsin (in the dark state), and magnetization-recovery plots (b) used to determine the spin-lattice (T_1_Z) relaxation time (T = −150 °C). One can observe much faster relaxation for the C13-Me than for C9-Me group, indicating the different environments for the retinylidene methyl groups in the rhodopsin binding pocket (8). Figure adapted from ref. (8).

Fig. 6

Fig. 6

Temperature dependences of the Zeeman (T_1_Z) relaxation times characterize the molecular dynamics of retinal ligand (8,10) in the dark and Meta II states. The slope of the plots directly provides the activation energy for methyl group spinning. The pre-exponential factors for motional parameters k, D||, and _D_⊥ (see text) can be determined by fitting the T_1_Z temperature dependences. Analysis of the relaxation data indicates relatively fast rotation of the C9-Me group, intermediate for the C13-Me group, and slow for the C5-Me group at low temperatures (Fig. 6), The rotational rates correlate with the corresponding activation energies Ea for these groups, and are possibly related to intra-retinal potential barriers (10). It is also shown that both 3-fold hops and rotational diffusion models give good agreement with the experimental T_1_Z relaxation times (8,10). Figure adapted from ref. (8).

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