Photoexcitation of Rhodopsin: Conformation Changes in the Chromophore, Protein and Associated Lipids as Determined by Ftir Difference Spectroscopy

DeGrip, Willem J.; Gray, D.; Gillespie, John A.; Bovee, P.H.M.; Berg, E. M. M. Van Den; Lugtenburg, J.; Rothschild, Kenneth J.

doi:10.1111/j.1751-1097.1988.tb02852.x

Cited by 69 publications

(67 citation statements)

References 49 publications

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“…The film is then rehydrated prior to insertion into a sealed transmittance cell as described previously (28). Transmission FTIR difference spectra of the hydrated rhodopsin films were recorded at 10°C using methods similar to those reported previously (29,30). Briefly, the H 2 O content of the sample was monitored by measuring the intensity ratio of the 3400-cm Ϫ1 band (O-H stretch mode) to the protein Amide II band near 1545 cm…”

Section: Methodsmentioning

confidence: 99%

“…DISCUSSION FTIR difference spectroscopy has been used extensively to study conformational changes in membrane proteins (42)(43)(44). In the case of rhodopsin, structural changes of the retinylidene chromophore and protonation and/or hydrogen bonding changes of Asp, Glu, and Cys residues have been detected at different stages of the photoactivation cascade (28,30,35,36,(45)(46)(47)(48)(49)(50)(51). In some cases, assignment of vibrational bands to individual amino acid residues in rhodopsin was facilitated by site-directed mutagenesis (28,35).…”

Section: Figurementioning

confidence: 99%

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Tyrosine Structural Changes Detected during the Photoactivation of Rhodopsin

DeLange¹,

Klaassen²,

Wallace-Williams³

et al. 1998

Journal of Biological Chemistry

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We present the first Fourier transform infrared (FTIR) analysis of an isotope-labeled eukaryotic membrane protein. A combination of isotope labeling and FTIR difference spectroscopy was used to investigate the possible involvement of tyrosines in the photoactivation of rhodopsin (Rho). Rho 3 MII difference spectra were obtained at 10°C for unlabeled recombinant Rho and isotope-labeled L-[ring-2 H 4 ]Tyr-Rho expressed in Spodoptera frugiperda cells grown on a stringent culture medium containing enriched L-[ring-2 H 4 ]Tyr and isolated using a His 6 tag. A comparison of these difference spectra revealed reproducible changes in bands that correspond to tyrosine and tyrosinate vibrational modes. A similar pattern of tyrosine/tyrosinate bands has also been observed in the bR 3 M transition in bacteriorhodopsin, although the sign of the bands is reversed. In bacteriorhodopsin, these bands were assigned to Tyr-185, which along with Pro-186 in the Fhelix, may form a hinge that facilitates ␣-helix movement.Elucidation of the mechanism of photoactivation of rhodopsin (Rho), 1 the light receptor in vision, remains an important problem in biology (1). Rhodopsin is an integral membrane protein found in the disc photoreceptor membranes of rod outer segments (2, 3) with a core structure consisting of seven transmembrane ␣-helices (4 -7). Upon light absorption, the rhodopsin chromophore, 11-cis-retinal, rapidly isomerizes to an alltrans configuration (8, 9) followed by a series of thermal transitions (Batho 3 Lumi 3 Meta I 3 Meta II) (10, 11). Signal transduction occurs upon formation of the Meta II intermediate, which binds and activates the G-protein transducin (12, 13). Because rhodopsin is a G-protein-coupled receptor, elucidation of its molecular mechanism is likely to be of general importance for the vast suprafamily of G-protein-coupled receptors, which include the ␤-adrenergic receptor (14, 15) and olfactory receptors (16).Thus far, bR is the only IMP with a seven-helix transmembrane motif whose structure has been elucidated at atomic resolution (17)(18)(19). This structure confirmed key features of an earlier "spectroscopic" model based in part on site-directed mutagenesis and FTIR difference spectroscopy (20 -23). For example, a retinal binding pocket was predicted on the basis of FTIR, UV-visible spectroscopy, and site-directed mutagenesis (20 -24), which is formed in part from several residues on the F-helix (helix 6 in rhodopsin), including two tryptophans (Trp-182, Trp-189), a proline (Pro-186), and a tyrosine (Tyr-185). These residues, along with several others from the C-helix (Trp-86, Thr-89, and Asp-85) are in close proximity to the retinal chromophore and act to constrain its structure in an all-trans configuration. In addition, these residues are in a good position to couple retinal isomerization to protein changes involved in proton transport, including a change in the structure and orientation of the F-helix.A comparable combination of Trp, Pro, and Tyr residues (WXPY) is fully conserved in helix 6 of all ...

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Section: Methodsmentioning

confidence: 99%

Section: Figurementioning

confidence: 99%

Tyrosine Structural Changes Detected during the Photoactivation of Rhodopsin

DeLange¹,

Klaassen²,

Wallace-Williams³

et al. 1998

Journal of Biological Chemistry

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“…This form of vibrational spectroscopy has been shown to be a powerful method for studying the molecular changes occurring in rhodopsin, from the formation of bathorhodopsin through the decay of MII (12)(13)(14)(15)(16). From previous experiments on bovine rhodopsin, it is known that three C=O stretching vibrations of membraneembedded protonated carboxyl groups are affected during the thermal relaxation to the MII state (12,(17)(18)(19).…”

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confidence: 99%

Protonation states of membrane-embedded carboxylic acid groups in rhodopsin and metarhodopsin II: a Fourier-transform infrared spectroscopy study of site-directed mutants.

Fahmy

Jäger²,

Beck³

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

258

290

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A method was developed to measure Fouriertransform infrared (FTIR) difference spectra of detergentsolubilized rhodopsin expressed in COS cells. Experiments were performed on native bovine rhodopsin, rhodopsin expressed in COS cells, and three expressed rhodopsin mutants with amino acid replacements of membrane-embedded carboxylic acid groups: Asp-83 -* Asn (D83N), Gln (E122Q), and the double mutant D83N/E122Q. Each of the mutant opsins bound 11-cis-retinal to yield a visible light-absorbing pigment. Upon illumination, each of the mutant pigments formed a metarhodopsin fl-like species with maximal absorption at 380 nm that was able to activate guanine nucleotide exchange by btanducin. Rhodopsin versus metarhodopsin iH-like photoproduct FTIR-difference spectra were recorded for each sample. The COS-ceil rhodopsin and mutant difference spectra showed close correspondence to that of rhodopsin from disc membranes.Difference bands (rhodopsin/metarhodopsin II) at 1767/1750 cm'i and at 1734/1745 cm-' were absent from the spectra of mutants D83N and E122Q, respectively. Both bands were absent from the spectrum of the double mutant D83N/E122Q. These results show that Asp-83 and Glu-122 are protonated both in rhodopsin and in metarhodopsin H, in agreement with the isotope effects observed in spectra measured in 2H20. A photoproduct band at 1712 cm-' was not affected by either single or double replacements at positions 83 and 122. We deduce that the 1712 cm-' band arises from the protonation of Glu-113 in metarhodopsin II. Rhodopsin is a member of the superfamily of seventransmembrane-helix, G protein-coupled receptors. The rhodopsin chromophore 11-cis-retinal is covalently bound to the protein via a protonated Schiff base linkage (1) to a lysine residue (Lys-296 in bovine rhodopsin) (2, 3). After photoisomerization of the chromophore, thermal relaxation leads to an active conformation, R*, which binds the G protein transducin and thereby couples photon absorption to the visual signal transduction cascade. It has been shown by chemical modifications of Lys-296 in bovine rhodopsin that the deprotonation of the Schiff base is a prerequisite for R* formation (4, 5). Spectroscopically, this state is designated metarhodopsin II (MII) and characterized by a visible absorption maximum (Am.) at 380 nm, indicative of the unprotonated Schiff base of all-trans-retinal. Biochemical studies (6-10) and resonance Raman spectroscopy (11) of recombinant rhodopsins have shown that the positive charge at the Schiff base nitrogen in rhodopsin is stabilized by Glu-113, which acts as a Schiff base counterion in the transmembrane domain of the opsin.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.To investigate the protonation states and possible protonation changes of membrane-embedded carboxyl groups in rhodopsin and its MII photoproduct, we have performed Fourier-transform i...

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“…The first intermediate, bathorhodopsin, has been extensively studied using FTIR spectroscopy (18)(19)(20)(21)(22) and exhibits protein changes such as carbonyl stretch shifts arising from E122 and D83 (21,22), changes in hydrogen bonding of several water molecules (22), alterations in cysteine S-H and backbone amide N-H stretches (18), a shift in the T118 O-H stretch (23), and modifications in amide I and amide II modes (20). A number of these changes are isomer-specific (18,19,21), indicating that direct protein-chromophore interactions may be responsible for the alterations.…”

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Picosecond Dynamics of G-Protein Coupled Receptor Activation in Rhodopsin from Time-Resolved UV Resonance Raman Spectroscopy

2003

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The protein response to retinal chromophore isomerization in the visual pigment rhodopsin is studied using picosecond time-resolved UV resonance Raman spectroscopy. High signal-to-noise Raman spectra are obtained using a 1 kHz Ti:Sapphire laser apparatus that provides <3 ps visible (466 nm) pump and UV (233 nm) probe pulses. When there is no time delay between the pump and probe events, tryptophan modes W18, W16, and W3 exhibit decreased Raman scattering intensity. At longer pump-probe time delays of +5 and +20 ps, both tryptophan (W18, W16, W3, and W1) and tyrosine (Y1 + 2xY16a, Y7a, Y8a) peak intensities drop by up to 3%. These intensity changes are attributed to decreased hydrophobicity in the microenvironment near at least one tryptophan and one tyrosine residue that likely arise from weakened interaction with the β-ionone ring of the chromophore following cis-to-trans isomerization. Examination of the crystal structure suggests that W265 and Y268 are responsible for these signals. These UV Raman spectral changes are nearly identical to those observed for the rhodopsinto-Meta I transition, implying that impulsively driven protein motion by the isomerizing chromophore during the 200 fs primary transition drives key structural changes that lead to protein activation.Knowledge of the initial response of a protein to an activating agent or event is required to derive a mechanistic understanding of protein function. In the case of the visual pigment rhodopsin, absorption of a single visible photon by the antagonist 11-cis retinal chromophore results in the formation of a highly energetic activated all-trans photo-product (1). This stored energy is transduced into global protein conformational changes at the Meta I-Meta II stages that trigger the G-protein visual cascade (2). The mechanism by which the photoisomerization reaction induces protein conformational changes is not understood, largely due to the lack of high temporal resolution structural tools that can elucidate these early events. Here, we report the first structural study of the protein conformational changes associated with the primary photochemical event in vision using picosecond time-resolved UV resonance Raman vibrational spectroscopy.Vibrational structural studies of biomolecules have greatly advanced since the introduction of UV resonance Raman (UVRR) 1 spectroscopy (3-5). When the excitation wavelength is tuned between ∼195 and 260 nm, strong resonance Raman scattering from the peptide backbone and aromatic amino acids provides vibrational information on local protein structure and environmental changes. This technique has been used, for example, to study rhodopsin protein structure (6), protein folding (7, 8), and protein-ligand interactions (9, 10). In addition, time- † We dedicate this paper to the memory of Helena (Ilona) Anna Palings (1956Palings ( -2002. This work was supported by grants from the National Institutes of Health (EY-02051) and the National Science Foundation (CHE-98-01651 (6,27,28), there remains a lack of understa...

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Photoexcitation of Rhodopsin: Conformation Changes in the Chromophore, Protein and Associated Lipids as Determined by Ftir Difference Spectroscopy

Cited by 69 publications

References 49 publications

Tyrosine Structural Changes Detected during the Photoactivation of Rhodopsin

Tyrosine Structural Changes Detected during the Photoactivation of Rhodopsin

Protonation states of membrane-embedded carboxylic acid groups in rhodopsin and metarhodopsin II: a Fourier-transform infrared spectroscopy study of site-directed mutants.

Picosecond Dynamics of G-Protein Coupled Receptor Activation in Rhodopsin from Time-Resolved UV Resonance Raman Spectroscopy

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