Chromophore Distortions in Photointermediates of Proteorhodopsin Visualized by Dynamic Nuclear Polarization-Enhanced Solid-State NMR (original) (raw)
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Journal of the American Chemical Society, 2014
Proteorhodopsins (PRs) found in marine microbes are the most abundant retinal-based photoreceptors on this planet. PR variants show high levels of environmental adaptation, as their colors are tuned to the optimal wavelength of available light. The two major green and blue subfamilies can be interconverted through a L/Q point mutation at position 105. Here we reveal the structural basis behind this intriguing color-tuning effect. High-field solid-state NMR spectroscopy was used to visualize structural changes within green PR directly within the lipid bilayer upon introduction of the green-blue L105Q mutation. The observed effects are localized within the binding pocket and close to retinal carbons C14 and C15. Subsequently, magic-angle spinning (MAS) NMR spectroscopy with sensitivity enhancement by dynamic nuclear polarization (DNP) was applied to determine precisely the retinal structure around C14-C15. Upon mutation, a significantly stretched C14-C15 bond, deshielding of C15, and ...
Photochemistry and Photobiology, 2009
Solid-state NMR spectroscopy gives a powerful avenue for investigating G protein-coupled receptors and other integral membrane proteins in a native-like environment. This article reviews the use of solid-state 2 H NMR to study the retinal cofactor of rhodopsin in the dark state as well as the meta I and meta II photointermediates. Site-specific 2 H NMR labels have been introduced into three regions (methyl groups) of retinal that are crucially important for the photochemical function of rhodopsin. Despite its phenomenal stability 2 H NMR spectroscopy indicates retinal undergoes rapid fluctuations within the protein binding cavity. The spectral lineshapes reveal the methyl groups spin rapidly about their three-fold (C 3 ) axes with an order parameter for the off-axial motion of S C3 % 0:9: For the dark state, the 2 H NMR structure of 11-cis-retinal manifests torsional twisting of both the polyene chain and the b-ionone ring due to steric interactions of the ligand and the protein. Retinal is accommodated within the rhodopsin binding pocket with a negative pretwist about the C11=C12 double bond. Conformational distortion explains its rapid photochemistry and reveals the trajectory of the 11-cis to trans isomerization. In addition, 2 H NMR has been applied to study the retinylidene dynamics in the dark and light-activated states. Upon isomerization there are drastic changes in the mobility of all three methyl groups. The relaxation data support an activation mechanism whereby the b-ionone ring of retinal stays in nearly the same environment, without a large displacement of the ligand. Interactions of the b-ionone ring and the retinylidene Schiff base with the protein transmit the force of the retinal isomerization. Solid-state 2 H NMR thus provides information about the flow of energy that triggers changes in hydrogen-bonding networks and helix movements in the activation mechanism of the photoreceptor. †This paper is part of the
2000
We present a solid-state NMR study of metarhodopsin-I, the pre-discharge intermediate of the photochemical signal transduction cascade of rhodopsin, which is the 41 kDa integral membrane protein that triggers phototransduction in vertebrate rod cells. The H-C10-C11-H torsional angles of the retinylidene chromophore in bovine rhodopsin and metarhodopsin-I were determined simultaneously in the photo-activated membrane-bound state, using doublequantum heteronuclear local field spectroscopy. The torsional angles were estimated to be |φ| = 160 ± 10 • for rhodopsin and φ = 180 ± 25 • for metarhodopsin-I. The result is consistent with current models of the photoinduced conformational transitions in the chromophore, in which the 11-Z retinal ground state is twisted, while the later photointermediates have a planar all-E conformation.
Proceedings of the National Academy of Sciences of the United States of America, 2008
By exploiting dynamic nuclear polarization (DNP) at 90 K, we observe the first NMR spectrum of the K intermediate in the ion-motive photocycle of bacteriorhodopsin. The intermediate is identified by its reversion to the resting state of the protein in red light and by its thermal decay to the L intermediate. The (15)N chemical shift of the Schiff base in K indicates that contact has been lost with its counterion. Under these circumstances, the visible absorption of K is expected to be more red-shifted than is observed and this suggests torsion around single bonds of the retinylidene chromophore. This is in contrast to the development of a strong counterion interaction and double bond torsion in L. Thus, photon energy is stored in electrostatic modes in K and is transferred to torsional modes in L. This transfer is facilitated by the reduction in bond alternation that occurs with the initial loss of the counterion interaction, and is driven by the attraction of the Schiff base to a n...
Solid-state NMR studies on the mechanism of the opsin shift in the visual pigment rhodopsin
Biochemistry, 1990
Solid-state 13C NMR spectra have been obtained of bovine rhodopsin and isorhodopsin regenerated with retinal selectively 13C labeled along the polyene chain. In rhodopsin, the chemical shifts for 13C-5, 13C-6, 13C-7,13C-14, and 13C-15 correspond closely to the chemical shifts observed in the 11-cis protonated Schiff base (PSB) model compound. Differences in chemical shift relative to the 11-cis PSB chloride salt are observed for positions 8 through 13, with the largest deshielding (6.2 ppm) localized at position 13. The localized deshielding at C-13 supports previous models of the opsin shift in rhodopsin that place a protein perturbation in the vicinity of position 13. Spectra obtained of isorhodopsin regenerated with 13C-labeled 9-c«-retinals reveal large perturbations at 13C-7 and I3C-13. The similar deshielding of the 13C-13 resonance in both pigments supports the presence of a protein perturbation near position 13. However, the chemical shifts at positions 7 and 12 in isorhodopsin are not analogous to those observed in rhodopsin and suggest that the binding site interactions near these positions are different for the two pigments. The implications of these results for the mechanism of the opsin shift in these proteins are discussed.
Journal of The American Chemical Society, 2004
We have obtained carbon-carbon bond length data for the functional retinylidene chromophore of rhodopsin, with a spatial resolution of 3 pm. The very high resolution was obtained by performing doublequantum solid-state NMR on a set of noncrystalline isotopically labelled bovine rhodopsin samples. We detected localized perturbations of the carbon-carbon bond lengths of the retinylidene chromophore. The observations are consistent with a model in which the positive charge of the protonated Schiff base penetrates into the polyene chain and partially concentrates around the C13 position. This coincides with the proximity of a water molecule located between the glutamate-181 and serine-186 residues of the second extracellular loop, which is folded back into the transmembrane region. These measurements support the hypothesis that the polar residues of the second extracellular loop and the associated water molecule assist the rapid selective photoisomerization of the retinylidene chromophore by stabilizing a partial positive charge in the center of the polyene chain.
Journal of Biomolecular NMR, 2012
Double-quantum magic-angle-spinning NMR experiments were performed on 11,12-13 C 2-retinylidenerhodopsin under illumination at low temperature, in order to characterize torsional angle changes at the C11-C12 photoisomerization site. The sample was illuminated in the NMR rotor at low temperature (*120 K) in order to trap the primary photointermediate, bathorhodopsin. The NMR data are consistent with a strong torsional twist of the HCCH moiety at the isomerization site. Although the HCCH torsional twist was determined to be at least 40°, it was not possible to quantify it more closely. The presence of a strong twist is in agreement with previous Raman observations. The energetic implications of this geometric distortion are discussed.
The Journal of Physical Chemistry B, 2007
Recent studies demonstrate that photoactive proteins can react within several picoseconds to photon absorption by their chromophores. Faster subpicosecond protein responses have been suggested to occur in rhodopsinlike proteins where retinal photoisomerization may impulsively drive structural changes in nearby protein groups. Here, we test this possibility by investigating the earliest protein structural changes occurring in proteorhodopsin (PR) using ultrafast transient infrared (TIR) spectroscopy with ∼200 fs time resolution combined with nonperturbing isotope labeling. PR is a recently discovered microbial rhodopsin similar to bacteriorhodopsin (BR) found in marine proteobacteria and functions as a proton pump. Vibrational bands in the retinal fingerprint (1175-1215 cm-1) and ethylenic stretching (1500-1570 cm-1) regions characteristic of all-trans to 13-cis chromophore isomerization and formation of a red-shifted photointermediate appear with a 500-700 fs time constant after photoexcitation. Bands characteristic of partial return to the ground state evolve with a 2.0-3.5 ps time constant. In addition, a negative band appears at 1548 cm-1 with a time constant of 500-700 fs, which on the basis of total-15 N and retinal C15D (retinal with a deuterium on carbon 15) isotope labeling is assigned to an amide II peptide backbone mode that shifts to near 1538 cm-1 concomitantly with chromophore isomerization. Our results demonstrate that one or more peptide backbone groups in PR respond with a time constant of 500-700 fs, almost coincident with the light-driven retinylidene chromophore isomerization. The protein changes we observe on a subpicosecond time scale may be involved in storage of the absorbed photon energy subsequently utilized for proton transport.
Enlightening the photoactive site of channelrhodopsin-2 by DNP-enhanced solid-state NMR spectroscopy
Proceedings of the National Academy of Sciences of the United States of America, 2015
Channelrhodopsin-2 from Chlamydomonas reinhardtii is a light-gated ion channel. Over recent years, this ion channel has attracted considerable interest because of its unparalleled role in optogenetic applications. However, despite considerable efforts, an understanding of how molecular events during the photocycle, including the retinal trans-cis isomerization and the deprotonation/reprotonation of the Schiff base, are coupled to the channel-opening mechanism remains elusive. To elucidate this question, changes of conformation and configuration of several photocycle and conducting/nonconducting states need to be determined at atomic resolution. Here, we show that such data can be obtained by solid-state NMR enhanced by dynamic nuclear polarization applied to (15)N-labeled channelrhodopsin-2 carrying 14,15-(13)C2 retinal reconstituted into lipid bilayers. In its dark state, a pure all-trans retinal conformation with a stretched C14-C15 bond and a significant out-of-plane twist of the...
Deuterium NMR Structure of Retinal in the Ground State of Rhodopsin †
Biochemistry, 2004
The conformation of retinal bound to the G protein-coupled receptor rhodopsin is intimately linked to its photochemistry, which initiates the visual process. Site-directed deuterium ( 2 H) NMR spectroscopy was used to investigate the structure of retinal within the binding pocket of bovine rhodopsin. Aligned recombinant membranes were studied containing rhodopsin that was regenerated with retinal 2 H-labeled at the C 5 , C 9 , or C 13 methyl groups by total synthesis. Studies were conducted at temperatures below the gel to liquid-crystalline phase transition of the membrane lipid bilayer, where rotational and translational diffusion of rhodopsin is effectively quenched. The experimental tilt series of 2 H NMR spectra were fit to a theoretical line shape analysis [Nevzorov, A. A., Moltke, S., Heyn, M. P., and Brown, M. F. (1999) J. Am. Chem. Soc. 121,[7636][7637][7638][7639][7640][7641][7642][7643] giving the retinylidene bond orientations with respect to the membrane normal in the dark state. Moreover, the relative orientations of pairs of methyl groups were used to calculate effective torsional angles between different planes of unsaturation of the retinal chromophore. Our results are consistent with significant conformational distortion of retinal, and they have important implications for quantum mechanical calculations of its electronic spectral properties. In particular, we find that the -ionone ring has a twisted 6-s-cis conformation, whereas the polyene chain is twisted 12-s-trans. The conformational strain of retinal as revealed by solid-state 2 H NMR is significant for explaining the quantum yields and mechanism of its ultrafast photoisomerization in visual pigments. This work provides a consensus view of the retinal conformation in rhodopsin as seen by X-ray diffraction, solid-state NMR spectroscopy, and quantum chemical calculations. mean square deviation; MNDO, modified neglect of diatomic overlap; TD, transition dipole.