Abstract
Rhodopsin is a prototype for the large Family A of G-protein–coupled receptors (GPCRs). These proteins regulate many signaling processes, and more than 35% of human pharmaceuticals are targeted against diseases related to dysfunctions of GPCR pathways. Membrane proteins such as GPCRs are challenging to crystallize for X-ray studies. In addition, their effective molar masses in detergent solutions push the limits for solution NMR spectroscopy. By contrast, solid-state NMR allows both the structure and dynamics of membrane proteins to be investigated in a natural lipid bilayer environment. Here, we describe solid-state 2H NMR methods for investigating structural and dynamical changes of the retinylidene cofactor of the GPCR rhodopsin upon photoillumination. Rhodopsin was regenerated with retinal containing 2H-labeled C5-, C9-, or C13-methyl groups. The receptor was recombined with phospholipid membranes, which were aligned on planar glass slides. The angular dependences of the 2H NMR spectra and the corresponding relaxation rates were measured for rhodopsin in the dark and in the cryo-trapped, preactive Meta-I and active Meta-II states. Analysis of the 2H NMR lineshapes using a static uniaxial distribution yields orientational restraints for the retinylidene conformation when bound to the protein. Solid-state 2H NMR relaxation data provide additional information on the motion of the bound cofactor. The structural and dynamical changes of retinal reveal how its functional groups (methyl groups and the β-ionone ring) affect rhodopsin light activation, and illustrate the opportunities of solid-state 2H NMR spectroscopy in studying membrane proteins.
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Keywords
- GPCRs
- Lipids
- Membrane proteins
- Membranes
- Molecular dynamics
- NMR relaxation
- NMR spectroscopy
- Rhodopsin activation
Membrane proteins comprise about one-third of the human genome and are responsible for regulating a variety of crucial cellular functions. Transport of proteins, small molecules, and ions across the membrane, reception of external stimuli and cellular signaling, membrane enzyme activity, and involvement in immune system function are among the crucial roles played by membrane proteins. Their location within biological membranes presents the greatest challenge limiting the techniques that can be used for studying such proteins [1]. For instance, the large size of the membrane proteins makes it difficult to use solution NMR spectroscopy; and growing high-quality membrane protein crystals as required for X-ray crystallographic studies is problematic. Advanced magnetic resonance techniques such as solid-state NMR spectroscopy thus become necessary for studying membrane proteins in a native-like lipid bilayer environment. Here, we present an application of solid-state NMR methods to membrane proteins using the visual receptor rhodopsin as the example.
Rhodopsin is the G-protein–coupled receptor responsible for the scotopic (dim light) vision of vertebrates. The chromophore 11-cis retinal acts as an inverse agonist, and locks the rhodopsin in the dark-state conformation. Following light absorption, it isomerizes to the all-trans form, releasing two ionic locks and rearranging hydrogen bonds that lead to movements of the transmembrane helices of the protein, yielding active rhodopsin. Clearly, the retinal chromophore is pivotal for maintaining the rhodopsin structure as well as its function. The temporal sequence of the photointermediates occurring during rhodopsin activation is illustrated in Fig. 1 [2, 3]. Most of these spectroscopically defined photointermediates are yet to be fully characterized, due to their transient nature, and they are trapped under various conditions such as temperature, pH, and lipid composition. The cognate G-protein of rhodopsin, transducin (Gt), is activated by the Meta-II state. The equilibrium between the inactive Meta-I and the active Meta-II species depends on hydration, pH, and the lipid bilayer composition, allowing these metastable species to be studied by varying the above conditions. Magnetic resonance methods such as electron paramagnetic resonance (EPR) [4], solid-state 13C NMR [5,6,7,8,9,10,11,12], and solid-state 2H NMR spectroscopy [13,14,15,16,17,18] together with X-ray techniques [19, 20] reveal crucial information about these intermediates in the activation of rhodopsin. Here, we review the use of solid-state 2H NMR spectroscopy to probe the structural and dynamical changes associated with rhodopsin activation in a natural lipid bilayer environment.
Structural Studies of Rhodopsin Using Solid-State Deuterium NMR Spectroscopy
Site-directed deuterium labeling of the retinal allows one to probe specific groups of the activating ligand. For these experiments, retinal is synthesized with 2H-labeled methyl groups at positions C5, C9, or C13, namely (11-Z-[5-C2H3]-retinal, 11-Z-[9-C2H3]-retinal, or 11-Z-[13-C2H3]-retinal) [17]. The deuterated retinal is incorporated into the binding pocket of opsin. The regenerated rhodopsin is then recombined with synthetic phospholipids to produce a native-like environment to study the structure and dynamics of the chromophore in various states of the receptor [16–18]. Isotopic labeling of each methyl group of 11-cis retinal allows one to probe different regions of the binding cavity, and identify changes during the activation of rhodopsin.
The chemical structure of 11-cis retinal (with the carbon atoms numbered) covalently bound to Lys296 of helix 7 (H7) of rhodopsin is provided in Fig. 2a. Illustrative deuterium NMR spectra of rhodopsin containing retinal with deuterated methyl groups at various positions (C5, C9, and C13) in POPC lipid membranes are shown in Fig. 2b–f. Aqueous dispersions of the recombinant membranes (unaligned) yield powder-type deuterium NMR spectra, where the Pake formula, Eq. (1) as explained in Ref. [21], is used for spectral simulation:
In the above formula p(ξ±) is the probability density, where ξ± ɛ [∓½, ±1] is the reduced frequency for each of the two spectral branches, and \( \tilde{\theta} \) is the overall angle of the C–C2H3 bond axis to the main B 0 magnetic field.
Use of the Pake formula and the spectral simulation provide the following information. The motion-averaged 2H NMR lineshapes yield residual quadrupolar couplings (RQCs) of 〈χQ〉 = 50.7–52.0 kHz. The occurrence of rapid spinning of the retinylidene methyl groups about their threefold axes is thus established, with rotational correlation times <10−5 s down to about −160 °C. Methyl-group order parameters of \( {S}_{C_3}\approx 0.9 \) are measured directly from the RQCs, amounting to ≈15° off-axis fluctuations. The dynamics entail methyl rotations with respect to the unsaturated polyene chain and the β-ionone ring, plus their reorientations within the binding pocket.
By aligning the POPC membranes containing rhodopsin on planar substrates, one can determine the average orientation of the C–C2H3 bond axis (principal axis of 2H quadrupolar coupling tensor, parallel to methyl threefold axis due to fast spinning) versus the membrane normal frame. The experimental arrangement (Fig. 3) entails a stack of supported membranes including rhodopsin with retinal 2H-labeled at the C5-, C9-, or C13-methyl groups. Rhodopsin is shown by the seven transmembrane helices (Fig. 3). At top right, the retinylidene ligand is depicted to illuminate the structure calculation (Fig. 3). A static uniaxial distribution is assumed, and the orientation θB of the C–C2H3 bond axis to the local membrane frame is established by calculating the 2H NMR lineshape versus the tilt angle θ between the average membrane normal n 0 and the main magnetic field B 0 (tilt series). For aligned samples, the simulation parameters entail the C–C2H3 bond orientation, the residual coupling 〈χQ〉, the mosaic spread σ, and the intrinsic line broadening.
Solid-state 2H NMR spectra in the dark state acquired at T = −150 °C are shown (Fig. 4a) for aligned rhodopsin/POPC (1:50) bilayers, with retinal 2H-labeled at the C5-, C9-, or C13-methyl groups, respectively. The results (Fig. 4a) were obtained for the θ = 0° orientation of the membrane normal to the magnetic field B 0 , where the lineshape is very sensitive to the methyl group orientation (θB). Similar experiments were conducted at temperatures of T = −60 and −30 °C. Notably, the solid-state 2H NMR spectra reveal only a minor temperature variation over a broad interval of 120 °C. Theoretical simulations are overlaid on the experimental 2H NMR data, and C–C2H3 bond orientations of 70 ± 3°, 52 ± 3°, and 68 ± 2° are found for the C5-, C9-, and C13-methyl groups of retinal in the rhodopsin dark state [16]. The data are explained by the average bond orientations and torsion angles of the retinylidene ligand bound to rhodopsin. Accurate bond orientations of the 2H-labeled methyl groups are determined, despite the appreciable alignment disorder (mosaic spread σ = 18–21°). Values for the alignment disorder are greater than in earlier studies of purple membranes containing bacteriorhodopsin [22], which may be due to rhodopsin (Mr = 40 kDa) being a larger molecule, together with the additional extramembranous regions [23]. These values differ from earlier work [24, 25]; yet they correspond well with X-ray studies of single rhodopsin crystals [26, 27].
Deuterium NMR Lineshapes
A simple analytical model can then be introduced (Fig. 4) for establishing the retinal conformation when bound to rhodopsin. Three planes of unsaturation are assumed, encompassing the β-ionone ring and the polyene chain to either side of the C12–C13 bond [16, 18, 28,29,30]. Relative orientations of the pairs of 2H-labeled methyl groups are used for calculating the effective dihedral angles for the various planes of unsaturation. By conducting studies of aligned samples, the 2H NMR data allow one to establish the orientation of the retinal cofactor bound to rhodopsin. Each molecular plane (here designated A, B, or C) is defined by two vectors, and thus two angular constraints are required to specify its orientation in space. The three planes have two bonds in common; hence, a total of four independent parameters (i.e., degrees of freedom) must be introduced to define the structure of the retinal molecule within its binding cavity. Because experimental data for the C1R,S methyl groups are currently unavailable, the electronic transition dipole moment of the retinylidene chromophore obtained from linear dichroism measurements can be used as a fourth orientational restraint [31,32,33,34].
It follows that the torsion angles χi,k between the A, B, and C planes are given by [16]:
In the above formula, \( {\theta}_{\mathrm{B}}^{(i)} \) and \( {\theta}_{\mathrm{B}}^{(k)} \) designate the orientations to the local membrane normal n of the various methyl groups within the consecutive planes. In addition, θ i and θ k are the angles of the two methyl axes to the C i –C k bond whose torsion angle is χi,k as in the case of the C6–C7 or the C12–C13 bond. Note that the C i –C k bond angle to the local membrane normal is \( {\theta}_{\mathrm{B}}^{\left(i,k\right)} \) and is obtained from the C9-methyl orientation, plus the transition dipole moment. For the torsion angles between the A, B, or C planes, multiple solutions are found (and multiple retinal geometries), which correspond to a single set of experimental methyl bond orientations. A total of 64 combinations for the relative orientations of the A, B, and C planes are possible for retinal bound to rhodopsin, giving 128 possible retinal configurations with the even parity taken into account (e.g., up and down orientation of the magnetic field cannot be distinguished). What is more, circular dichroism (CD) data [29, 35] and carbon–carbon distances obtained from rotational resonance solid-state 13C NMR studies [12, 36] can be introduced. It is then possible to obtain a simple physical result, in analogy to solution NMR spectroscopy.
According to these procedures, the angle between planes B and C is established using Eq. (2) to be χ9,13 = +150 ± 4° (+147 ± 4°) (Fig. 4b). The two values correspond to a C11=C12–C13 bond angle of 120 or 130°, respectively [15]. Note also that distortion of the C11=C12–C13 bond angle from ideal orbital geometry may take place due to steric hindrance of the C13-methyl group with the retinal hydrogen H10, or it may be induced by the rhodopsin binding pocket. Considering next the β-ionone ring, introducing positional restraints for the C8-to-C18 and C8-to-C16/C17 distances from solid-state 13C NMR [36] plus the chirality known from CD studies [35], the C6–C7 dihedral angle of χ5,9 = −65 ± 6° is the physical solution. It represents a negatively twisted 6-s-cis conformation (Fig. 4b). To further specify the 11-cis-retinylidene structure derived from solid-state 2H NMR, it was inserted into the binding pocket of the rhodopsin X-ray structure (resolution of 2.2 Å; PDB accession code 1U19) [27]. The Schiff base end of retinal was superimposed with the Lys296 nitrogen, keeping the C12 to C15 carbon atoms near to the X-ray coordinates. Even so, a simple three-plane model did not fit into the binding pocket, because of the multiple steric clashes of retinal with the Tyr178 and Cys187 side chains of extracellular loop E2, as well as Met207, Tyr268, and Ala292 in helices H5, H6, and H7, respectively. To resolve this difficulty, an additional twist of the C11=C12 bond of the polyene chain was assumed [16, 37]. Adopting an extended three-plane model, the C11=C12 dihedral angle was established by fitting the C10-to-C20 and C11-to-C20 distances to 13C NMR rotational resonance data [12]. As a result, the C11=C12 torsion angle was found to be −25 ± 10° and the C12–C13 torsion angle 176 ± 6°, where the twist is predominantly localized to the C11=C12 bond. Note that the C10-to-C20 and C11-to-C20 distances are less sensitive to the C11=C12 torsion angle. The structure deduced for retinal (Fig. 4c) in this way corresponds to the rhodopsin N-terminus at the top (extracellular side), and the C-terminus at the bottom (cytoplasmic side).
To summarize at this point using solid-state 2H NMR spectroscopy, the structural studies reveal twisting of the retinal chromophore when bound to rhodopsin in the dark state. This finding is in accord with other biophysical studies [18, 26, 29, 30, 34, 36,37,38,39,40,41,42,43,44,45] and is key to understanding its photoreaction dynamics [46,47,48,49]. Of notable significance for rhodopsin is the torsional twisting about the C11=C12 double bond, together with the twist of the β-ionone ring [30]. The finding of a nonplanar structure of the retinal ligand is consistent with the presence of hydrogen-out-of-plane (HOOP) modes in resonance Raman [47, 49,50,51] and FTIR [40] spectroscopies and with circular dichroism (CD) studies [29, 38, 42, 43]. All of these features are significant to explaining how release of the photonic energy is used to initiate visual signaling by rhodopsin in the natural photoreceptor membranes [16, 29, 31, 36].
In a manner similar to the above studies, the Meta-I state can also be cryo-trapped in planar-supported bilayers to determine the changes in the retinal conformation and orientation induced by light absorption. Solid-state 2H NMR spectra acquired at −100 °C for macroscopically aligned POPC membranes at θ = 0° tilt angle allow comparison of the dark state (Fig. 4a–c) to the Meta-I state (Fig. 4d–f). For the bound retinylidene ligand, the greatest differences are found for the C9–C2H3 group, with smaller variations for the C5–C2H3 and C13–C2H3 groups (Fig. 4d). These differences in the 2H NMR spectra manifest the bond orientation θB as well as the alignment disorder (mosaic spread σ). In the preactive Meta-I state, orientations are determined for the C5-, C9-, and C13-methyl groups θB of 72 ± 4°, 53 ± 3°, and 59 ± 3°, respectively. Because of the larger mosaic spread of Meta-I (σ = 22–25°) versus dark-state rhodopsin (σ = 18–21°), the 2H NMR spectra for the C9-methyl differ, although the bond orientations are similar (Fig. 4d). In contrast, for the C13-methyl group, a smaller spectral difference is evident with a larger change in bond orientation. Analogous to dark-state rhodopsin, the solid-state 2H NMR spectra establish that the retinylidene methyl substituents have rapid threefold rotation, with order parameters for off-axial motions of ≈0.9 within the Meta-I binding cavity (Fig. 4d).
Theoretical simulation of the solid-state 2H NMR spectra [21] provides the methyl group angles, which together with the electronic transition dipole moment define the orientations of the molecular fragments. The fragments are then assembled to yield to the retinal structure when bound to the protein. Examples (two for each plane) show that multiple solutions are possible for the retinal structure (Fig. 4e). By use of Eq. (2), and including experimental rotational resonance 13C NMR data [12] for the C10-to-C20 and C11-to-C20 carbon distances, the torsion angle between the B and C planes of χ9/13 = ±173 ± 4° is found. An almost fully relaxed all-trans conformation occurs in the preactive Meta-I state [11, 12]. What is more, solutions for the β-ionone ring of χ5/9 = ±32 and ±57° are found as two possibilities. For the Meta-I state, the region of the polyene chain with the C13-methyl group rotates about the С11=С12 double bond toward the membrane extracellular side. Evidently, the polyene chain becomes straightened, whereby the β-ionone ring is displaced along the retinal axis toward helix H5. The calculated Meta-I structures are further restrained by placing them into the dark-state rhodopsin X-ray structure [27]. Assuming the shape of the retinal binding cavity in Meta-I resembles the dark state, most of the retinal structures were eliminated because of the different position of the β-ionone ring in the binding pocket. The exception is the retinal structure with χ5/9 = −32° and χ9/13 = 173° (left of Fig. 4f) and the structure with χ5/9 = 32° and χ9/13 = −173° obtained by reflection (mirror symmetry transformation) in a vertical plane. Even so, the first structure fits best within the binding cavity, the only close contact being with the side chain of Trp265. By contrast, the mirror structure makes close contacts with the side groups of Glu122, Trp265, Tyr268, and Ala292, and thus it is less probable.
Retinal Molecular Dynamics and Solid-State 2H NMR Relaxation
Turning next to the retinal dynamics, in Fig. 5 the experimental 2H NMR relaxation experiments are summarized for rhodopsin in the dark state. Arrhenius-type plots of both the T1Z relaxation times and T1Q relaxation times are shown versus reciprocal temperature, for those methyl groups of the bound retinal crucial to its function. One can perceive that the structural fluctuations of retinal in the dark state are unaltered by phase transitions of the lipids or by the freezing of water. The retinal cofactor is deeply buried within the receptor core (Fig. 3), and hence the relaxation data can be smoothly extrapolated to physiological temperature. For the retinylidene C5-Me group (Fig. 5a, b), a clear minimum in T1Z is seen at −120 °C and for T1Q at −135 °C. By contrast, for the C9- and C13-Me groups, the T1Z and T1Q minima occur at much lower temperatures, outside the measured range (Fig. 5c, d) [52]. According to these results, site-specific differences exist in the dynamics of the retinylidene methyl groups when bound to rhodopsin, despite that the order parameters show little variation.
As discussed above, for slower motions, the T1Z and T1Q minima occur at higher temperature (to the left). Conversely, for faster motions, the minima occur at lower temperature (to the right) [13]. At a given minimum, the effective correlation time τ c is matched to the Larmor (resonance) frequency ω0 by τ c ≈ 1/ω0 = 13 ns. Moreover, the correlation times are less at higher temperatures, as observed for the different retinylidene methyl groups. The comparatively short T1Z and T1Q relaxation times for the C5-Me group indicate a mainly 6-s-cis conformation of the β-ionone ring [18], in contrast to 6-s-trans. (For a 6-s-cis conformation, the nonbonded (1,7) interactions with hydrogen H8 would give slower spinning of the C5-Me group versus (1,6) interactions with hydrogen H7 in the 6-s-trans conformation.) What is more, the polyene C9- and C13-methyl groups have longer T1Z times. They indicate greater mobility, because of the weak noncovalent interactions within the retinal ligand, and with the adjacent amino acid residues.
Generalized Model-Free Analysis for Nuclear Spin Relaxation Rates
At the initial stage, our approach [53] is evidently model free, because we directly measure the RQCs and derived C–C2H3 order parameters, plus the corresponding R1Z relaxation rates. In the next level of analysis, the theory of nuclear spin relaxation is introduced to investigate the molecular dynamics [53]. Experimentally, the nuclear spin-lattice relaxation rates depend on thermal motions of the C–2H bond (electric field gradient, EFG tensor) near the ω0 resonance frequency, and read [53]:
and
In the above expressions, T1Z is the relaxation time for Zeeman order and T1Q is that for quadrupolar order; χQ is the quadrupolar coupling constant; and ω0 is the nuclear resonance (Larmor) frequency. The spectral densities of motion are denoted by J1(ω0) and J2(2ω0) and describe the power of the fluctuations at ω0 and twice ω0. A simple physical picture is that matching of the power spectrum of the fluctuations to the Zeeman energy level gap drives the transitions between states of different nuclear spin angular momentum [53].
The general theory of NMR relaxation [53] can then be applied to analyze in greater detail the molecular dynamics of retinal bound to rhodopsin. Generalized model-free (GMF) analysis [53] describes how the irreducible spectral densities J m (ω) depend on the average methyl angle to the magnetic field, plus the mean-square amplitudes and rates of the motions. The spectral densities are given by:
where ω = mω0 and m = 1,2 [54]. Here, D(2)(Ω ij ) indicates the second-rank Wigner rotation matrix, and Ω ij ≡ (α ij , β ij , γ ij ) are the Euler angles [53] for the relative orientations of the coordinate systems {i, j} ≡ {P, I, M, L} under consideration (see Fig. 3). In this notation P is the principal axis system for the electric field gradient tensor of the C–2H bond, I specifies the instantaneous orientation of the methyl group, M the average orientation, and L the laboratory (B 0 ) axis system. Notably, the mean-square amplitudes are contained in the angular brackets and denote a time-ensemble average. The corresponding reduced spectral densities are indicated by \( {j}_{rq}^{(2)}\left(\omega \right)=2{\tau}_{rq}/\left(1+{\omega}^2{\tau_{rq}}^2\right) \), where τ rq are the rotational correlation times.
In the case of either membrane proteins [55] or lipids, using the GMF approach, the mean-square amplitudes are quantified by the segmental order parameters, and the rates by the motional correlation times [53]. For example, for a methyl group with Euler angles ΩPI = (0°, 70.5°, 0°), assuming ideal geometry, an effective coupling constant of \( {\chi}_{\mathrm{Q}}^{\mathrm{eff}}={\chi}_{\mathrm{Q}}{D}_{00}^{(2)}\left({\Omega}_{\mathrm{PI}}\right)=\hbox{--} \frac{1}{3}{\chi}_{\mathrm{Q}} \) is obtained. The order parameter of the threefold axis describes the off-axial fluctuations, and it is defined by \( {S}_{C_3}=\frac{1}{2}\left\langle 3{\cos}^2{\beta}_{\mathrm{IM}}\hbox{--} 1\right\rangle \), where βIM is the angle of the methyl orientation at any instant to its average value. Further theoretical analysis entails the introduction of a motional model [53, 56] to explain the fluctuations.
Applying the above formalism to rhodopsin, the T1Z times can be analyzed using either a 3-site jump model or a continuous diffusion model for rotation of the retinylidene methyl groups [53]. For N-fold jumps with a rate constant k about a single axis, the correlation times are [54, 56] 1/τ rq → 1/τ r = 4ksin2(πr/N) leading to 1/τ r = 3k for a methyl group. Alternatively, the limit of rotational diffusion about an axis can be considered. For continuous diffusion within a potential of mean torque [57], the rotational correlation times read:
Here, the symbols D∣∣ and D⊥ indicate the axial and off-axial diffusion coefficients for methyl rotation; and the moments μ rq and mean-squared moduli \( \left\langle {\left|{D}_{rq}^{(2)}\left({\Omega}_{\mathrm{IM}}\right)\right|}^2\right\rangle \) depend on both second- and fourth-rank order parameters [57]. For a strong-collision approximation, the correlation times are those of a rigid rotor [53]: 1/τ rq → 1/τ r = 6D⊥ + (D∣∣ − D⊥)r2. The limit of continuous diffusion, e.g., for diffusion about a single axis, 1/τ r = D∥r2 giving the results in Refs. [53, 56]. Finally, application of transition-state theory indicates the correlation times are inversely related to A exp(−Ea/RT), where A is the pre-exponential factor (k0 or D0) and Ea is the activation barrier (energy) for methyl rotation. For a rotational diffusion model D∣∣ = D0∣∣ exp(–Ea∣∣/RT) and D⊥ = D0⊥ exp(–Ea⊥/RT) and analogously for a threefold jump model [13, 53, 54, 57].
Retinal Dynamics in Inactive and Active Rhodopsin
As a next step, one is able to use NMR relaxation to investigate the changes in the dynamics of the retinal ligand of rhodopsin that occur upon light absorption. Compared to the dark state, pronounced T1Z differences are found in the Meta-I and Meta-II states that encapsulate the light-induced changes in the retinylidene dynamics (Fig. 6). The short T1Z relaxation times measured for the C5-methyl group in the dark state (Fig. 6a) indicate the β-ionone ring has a predominantly 6-s-cis conformation [13, 37], as opposed to 6-s-trans for bacteriorhodopsin [58]. Evidently [59], the β-ionone ring retains nearly the same local environment, and is not strongly affected by transitions among the dark, Meta-I, and Meta-II states (Fig. 6b, c) [37]. A shifting of the T1Z minimum for the C5-methyl to lower temperatures in Meta-II (Fig. 6c) indicates a reduction in the local correlation time, e.g., due to lowering Ea by changing the C6–C7 torsional angle and/or enhancing the ring mobility. For the functionally critical C9-Me group [60], a striking change in T1Z is evident in the Meta-I and Meta-II states, due to an increase in Ea for methyl spinning within the rhodopsin-binding pocket. What is more, similar Ea barriers of the C9- and C13-methyl groups upon 11-cis to all-trans isomerization are plausible, because they are on the same side of the retinal polyene chain (Figs. 6b, c).
Application of transition state theory allows the NMR relaxation rates to be quantified in terms of the activation barriers for the motions, together with the corresponding pre-exponential factors. Using transition state theory, large site-specific differences are found in the pre-exponential factors and activation barriers (Ea) (Fig. 7). For the C5-methyl substituent of the β-ionone ring (Ea = 10–15 kJ mol−1), the barrier values exceed those of the other methyl groups in dark, Meta-I, and Meta-II states. For the C5-methyl group of the retinylidene ligand, the relatively high activation energy Ea (calculated from the slope of the temperature dependences of the T1Z and T1Q relaxation times) of ≈12 kJ mol−1 is usual for methyl rotations in organic solids. Steric hindrance can occur because of nonbonded, intraretinal interactions with the polyene H8 hydrogen, and/or steric clashes with amino acid residues within the binding cavity, e.g., Glu122 or possibly Trp265. By contrast, the notably low Ea value (barrier) for the C9-methyl is most likely due to compensation of intraretinal steric interactions, as discussed below.
Quantum mechanical calculations for retinal model compounds [61, 62] confirm that the vanishingly small barrier to rotation of the C9-methyl group (Ea = 2 kJ mol−1 in the dark state) (Fig. 7a) is due to nonbonded (1,6) interactions with hydrogen atoms H7 and H11 of the polyene chain (see Fig. 1). In Fig. 7a, a simple explanation of why the C9-methyl group of retinal has a smaller activation energy than expected from quantum mechanical calculations for typical organic compounds is given [37, 61]. Hydrogen atoms H7 and H11 of the polyene chain have intraretinal (1,6) interactions that are approximated by a threefold rotational potential. These rotational potentials are in antiphase due to periodic eclipsing interactions; that is to say, they are offset by ≈60° giving a shallow resultant potential (Fig. 7a). Within the binding cavity, interactions with other residues are comparatively weak, because the C9-methyl is located in the space between Tyr268 and Thr118 in the crystal structure. By contrast, for the C13-methyl group near the protonated Schiff base (PSB) end of retinal (Fig. 7b), and the C5-methyl group of the β-ionone ring at the opposite end (Fig. 7c), the mobility is less because of nonbonded (1,7) interactions with hydrogens H10 and H8, respectively. For the C13-methyl group, the intermediate Ea barrier may be due to steric interactions with retinal hydrogen H10, and possibly Tyr268 as well.
Following light-induced isomerization of the bound retinal ligand, the most pronounced changes entail the polyene chain of retinal, rather than the β-ionone ring. In the sequence, dark to Meta-I to Meta-II, there is a systematic increase in the activation energy barrier (Ea) for the C9-methyl of the polyene chain, together with an opposite decrease for the C13-methyl substituent (Fig. 7d). For example, the activation barrier for the C9-methyl group rotation is increased about twofold; hence, its rotational dynamics become slower. Surprisingly, however, the C9-methyl group – which is essential to rhodopsin function [60] – remains as a dynamical hotspot. Following 11-cis to trans isomerization (Fig. 1), the (1,6) interactions of the C13-methyl group occur with both polyene hydrogens H11 and H15; hence, the C13-methyl barrier (Ea = 3–5 kJ mol−1) becomes similar to the C9-methyl barrier in the Meta-I and Meta-II states (Fig. 7d). Perhaps most striking, the C5-methyl barrier stays almost unchanged up to the active Meta-II state (Fig. 7d), in agreement with a mainly 6-s-cis conformation of the β-ionone ring [13, 37].
Challenges Ahead: Molecular Mechanism of Rhodopsin Activation
Obviously, many important details of the activation mechanism of rhodopsin in a natural membrane environment remain unsolved. Because the secondary and tertiary structures of the Rhodopsin (Family A) GPCRs are similar, the local interactions are expected to be crucially important for targeted drug design. For rhodopsin, despite that several X-ray crystal structures including Meta-II-like structures are available, the orientation of the chromophore is still uncertain. What is more, there is a controversy between X-ray and solid-state 13C NMR results. Notably, the retinal conformation in the active Meta-II state needs to be established with a high degree of certainty [63, 64]. The molecular dynamics of the retinal ligand and the rhodopsin overall structure in the activation mechanism are very important in the receptor activation, and probably other GPCRs as well. For studying GPCRs, a combination of different techniques is salutary, including molecular simulation methods that connect snapshots provided by X-ray and NMR studies of rhodopsin [52, 64], both in crystals as well as a natural lipid membrane environment.
Our current understanding of rhodopsin activation by retinal [13, 37] (Fig. 8) allows us to propose how local (ps–ns) motions of the ligand initiate the large-scale (ms) functional protein motions (Fig. 8a) [65–67]. In the inactive dark state, the low energy barrier to rotations of the C9-methyl group signifies a lack of steric clashes, because it occupies a slot between Tyr268 and Thr118. The C9-methyl group acts effectively as a hinge point for retinal isomerization, causing the C13-methyl and the C=NH+ groups to change their positions upon light activation (Fig. 8a). In the Meta-I NMR structure of retinal [13, 15, 16, 37], rotation of the C13-methyl shifts the β4 strand of the E2 loop upward to the extracellular (e) side (Fig. 8b). A hydrogen-bonding network involving TM helices H4–H6 is disrupted, as implied by a decrease in the rotational barrier (Ea) for the C13-methyl group. At the other end of the retinal ligand, the β-ionone ring is shifted toward the helix H5. A closer packing for the C5-methyl is suggested by the comparatively high Ea value in the Meta-I state (Fig. 7d). The first ionic lock of the retinylidene PSB on helix H7 with its complex counterion due to Glu113 (H3) and Glu181 (E2) is broken by internal proton transfer from the PSB to Glu113 in Meta-II. Elongation of retinal displaces the β-ionone ring away from Trp265 (H6) toward the H5–H6 helical interface [37, 63]. Initial tilting of helix H6 away from the H1–H4 helical core is accompanied by restructuring of helix H5, bringing Tyr223 closer to Arg135 and Gly231 near to Glu247 (Fig. 8c) [66]. This destabilizes the second ionic lock involving Glu134 and Arg135 of the conserved E(D)RY sequence of helix H3 plus Glu247 of helix H6 [66], transiently exposing the recognition sites for transducin (Gt) on the cytoplasmic face (c-side) (Fig. 8c) [67]. In the above model, rhodopsin activation entails collective movements of transmembrane helices H5 and H6, together with the cytoplasmic loops in the Meta-I to Meta-II equilibrium. The reversible helix fluctuations [65, 67] happen at kHz frequencies, matching the catalytic turnover rate for Gt binding and activation by rhodopsin [68]. Receptor activation thus entails a fluctuating equilibrium among states and substates on the energy landscape [13, 37]. A key question for future research is whether one can quantitatively trap a single activated rhodopsin species, or whether the receptor activation requires the dynamics of a conformational ensemble unlocked by photoisomerization of the retinal ligand.
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Perera, S.M.D.C., Xu, X., Molugu, T.R., Struts, A.V., Brown, M.F. (2018). Solid-State Deuterium NMR Spectroscopy of Rhodopsin. In: Webb, G. (eds) Modern Magnetic Resonance. Springer, Cham. https://doi.org/10.1007/978-3-319-28388-3_144
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