Photoactivation of rhodopsin and interaction with transducin in detergent micelles

König, Bernd; Welte, Wolfram; Hofmann, Klaus Peter

doi:10.1016/0014-5793(89)81811-3

Cited by 51 publications

(31 citation statements)

References 19 publications

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“…Thus dodecyl maltose, a mild detergent that dissolves the membrane, was added to reduce light scattering and greatly improve the clarity of the optical spectra. However, the presence of dodecyl maltose increases the rate of the MI 3 MII reaction and shifts the MI/MII equilibrium strongly toward metarhodopsin-II (55). Under the conditions of this experiment (pH 7.2, 4°C, 2.0 mM dodecyl maltoside) the absorbance components show that the equilibrium ratio of MII/MI is 1.2 (accounting for the higher extinction coefficient of MI).…”

Section: Conformational States Of Rhodopsin and The Activity Of K42-41l-mentioning

confidence: 66%

Equilibrium between Metarhodopsin-I and Metarhodopsin-II Is Dependent on the Conformation of the Third Cytoplasmic Loop

Piscitelli

Angel

Bailey

et al. 2006

Journal of Biological Chemistry

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Rhodopsin is a G-protein-coupled receptor (GPCR) that is the light detector in the rod cells of the eye. Rhodopsin is the best understood member of the large GPCR superfamily and is the only GPCR for which atomic resolution structures have been determined. However, these structures are for the inactive, dark-adapted form. Characterization of the conformational changes in rhodopsin caused by light-induced activation is of wide importance, because the metarhodopsin-II photoproduct is analogous to the agonist-occupied conformation of other GPCRs, and metarhodopsin-I may be similar to antagonist-occupied GPCR conformations. In this work we characterize the interaction of antibody K42-41L with the metarhodopsin photoproducts. K42-41L is shown to inhibit formation of metarhodopsin-II while it stabilizes the metarhodopsin-I state. Thus, K42-41L recognizes an epitope accessible in dark-adapted rhodopsin and metarhodopsin-I that is lost upon formation of metarhodopsin-II. Previous work has shown that the peptide TGALQERSK is able to mimic the K42-41L epitope, and we have now determined the structure of the K42-41L-peptide complex. The structure demonstrates a central role for elements of the rhodopsin C3 loop, particularly Gln 238 and Glu 239 , in the interaction with K42-41L. Geometric constraints taken from the antibody-bound peptide were used to model the epitope on the rhodopsin surface. The resulting model suggests that K42-41L locks the C3 loop into an extended conformation that is intermediate between two compact conformations seen in crystal structures of dark-adapted rhodopsin. Together, the structural and functional data strongly suggest that the equilibrium between metarhodopsin-I and metarhodopsin-II is dependent upon the conformation of the C3 loop. The biological implications of this model and its possible relations to dimeric and multimeric complexes of rhodopsin are discussed.Rhodopsin is a G-protein-coupled receptor (GPCR) 2 characterized by seven transmembrane helices (H1 through H7) (1). It is an efficient photoreceptor, with the chromophoric retinal cofactor undergoing photoisomerization from 11-cis to all-trans upon activation. The retinal isomerization propagates conformational changes through the lightexcited rhodopsin protein to a meta-stable active state, metarhodopsin-II, which is in equilibrium with the inactive metarhodopsin-I precursor (see Fig. 1A). Metarhodopsin-II binds to the heterotrimeric G-protein transducin (Gt) and triggers GDP release followed by GTP uptake (2, 3).A large fraction of existing drugs and drugs under development target GPCRs (4). The rhodopsin system is considered a prototypical model for the GPCR superfamily, and recent work has produced new insights on the biochemistry of rhodopsin (1, 2). However, many questions remain unanswered, particularly with respect to the mechanism of receptor and G-protein activation.Metarhodopsin-II is characterized by its specific ability to bind and activate multiple copies of the heterotrimeric G-protein transducin (Gt ␣␤␥ ) in rapid s...

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Section: Conformational States Of Rhodopsin and The Activity Of K42-41l-mentioning

confidence: 66%

Equilibrium between Metarhodopsin-I and Metarhodopsin-II Is Dependent on the Conformation of the Third Cytoplasmic Loop

Piscitelli

Angel

Bailey

et al. 2006

Journal of Biological Chemistry

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“…The MUM11 equilibrium has been shown to be sensitive to temperature, pH (Matthews et al, 1963) and pressure (Lamola et a]., 1974 ; Attwood and Gutfreund, 1980). Environmental properties, such as lipid composition, membrane fluidity (Applebury et al, 1974;DeGrip et al, 1983;Wiedmann et al, 1988;Mitchell et al, 1990;Gibson et a]., 1993;Brown, 1994), the degree of hydration (Rafferty and Shichi, 1981;Ganter et al, 1988), and the presence of detergents (Lamola et al, 1974;Konig et al, 1989) also affect the equilibrium. Furthermore, several mutations involving polar amino acid residues in rhodopsin have been reported that shift the MIMII equilibrium Nathans, 1992, 1993;DeCaluwC et al, 1995).…”

mentioning

confidence: 99%

“…Salt effects on the kinetics of rhodopsin bleaching have been reported for rhodopsin in detergent solution (Arnis and Hofmann, 1993). However, since almost all detergents dramatically alter the pH dependence of the MUM11 equilibrium (Matthews et al, 1963;Lamola et al, 1974;Konig et al, 1989), effects of ionic strengh on the apparent pK., of this equilibrium have not been investigated. We have analyzed these aspects using ultraviolet/visible and Fourier-transform infrared (FTIR) spectroscopies on the native rod-outer-segment (ROS) membrane.…”

mentioning

confidence: 99%

Modulation of the Metarhodopsin I/Metarhodopsin II Equilibrium of Bovine Rhodopsin by Ionic Strength

DeLange

Merkx

Bovée-Geurts

et al. 1997

European Journal of Biochemistry

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The effects of ionic strength on formation and decay of metarhodopsin I1 (MII), the active photointermediate of bovine rhodopsin, were studied in the native membrane environment by means of ultraviolet/ visible and Fourier-transform infrared (FTIR) spectroscopy. By increasing the concentration of KCI in the range from hypotonic to 4 M, the apparent pK, of the metarhodopsin I(MI)/MII equilibrium is shifted by approximately pH three, in favor of the MI1 intermediate. In addition, the apparent rate of MI1 formation is enhanced by an increase in ionic strength (about twofold in the presence of 2 M KCI). MI1 decay is independent of the salt concentration. Attenuated-total-reflectance/lTIR data show that the high-salt conditions have no effect on the rigidity of the membrane matrix and do not induce structural changes in the intermediates themselves. Different salts were tested for their ability to shift the MI/MII equilibrium; however, no clear ion dependence was observed. We interpret these results as an indication for direct involvement of the cytosolic surface charge in the regulation of the photochemical activity of bovine rhodopsi n.Keywords: bovine rhodopsin ; metarhodopsin hetarhodopsin I1 equilibrium ; pKt, ; ionic strength ; surface charge Rhodopsin is the photoreceptor protein located in the disk membranes of retinal rod photoreceptor cells, and is considered to be a model for the superfamily of guanine-nucleotide-binding regulatory protein (G protein)-coupled receptors (DeGrip et al., 1988;Hargrave and McDowell, 1992). It consists of the apoprotein opsin and the chromophore 1 1-cis-retinal, which is covalently linked through a Schiff base to Lys296 in bovine opsin. Photoexcitation of rhodopsin involves the rapid photoisomerization of the chromophore to the all-trans configuration. This primary photochemical event triggers a cascade of photointermediates involving a series of slower thermal transitions in the protein moiety. Under physiological conditions, the active metarhodopsin I1 intermediate (MII) is formed on a millisecond timescale (Emeis et al., 1982;Kibelbek et al., 1991). MI1 can bind and activate the G protein transducin, which ultimately leads to hyperpolarization of the rod photoreceptor cell and further neuronal transduction of the signal.MI1 is in equilibrium with its precursor metarhodopsin I (MI; Matthews et al., 1963). Therefore, to clarify the mechanism of visual excitation on a molecular level, understanding the factors that control the MUM11 transition is essential. The main characteristics of the MI/MII transition are considered to be (a) deprotonation of the retinal Schiff base and a net proton uptakeCorresponderzcr to

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“…They accelerate the MII absorption change by two orders of magnitude, as compared to the native membrane environment or digitonin (18). If proton uptake is obligatory for the MII conformation change, it must be accelerated as well and maintain its kinetic match to MII formation.…”

mentioning

confidence: 99%

“…It has been related to a free volume change in the hydrophobic space (18,20), as symbolized in Fig. 6 by the lateral expansion.…”

mentioning

confidence: 99%

Two different forms of metarhodopsin II: Schiff base deprotonation precedes proton uptake and signaling state.

Arnis

Hofmann

1993

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

174

164

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Rhodopsin is a retinal protein and a G-protein-coupled receptor; it shares with both of these families the seven helix structure. To generate the G-interacting helix-oop conformation, generally identified with the 380-nm absorbing metarhodopsin U (MU) photoproduct, the retinal Schiff base bond to the apoprotein must be deprotonated. This occurs as a key event also in the related retinal proteins, sensory rhodopsins, and the proton pump bacteriorhodopsin. In MUI, proton uptake from the aqueous phase must be involved as well, since its formation increases the pH of the aqueous medium and is accelerated under acidic conditions. In the native membrane, the pH effect matches MU formation kinetically, sugging that intramolecular and aqueous protonation changes contribute in concert to the protein transformation.We show here, however, that proton uptake, as indicated by bromocresol purple, and Schiff base deprotonation (380-nm absorption change) show different kinetics when the protein is solubilized in suitable detergents. Our data are consistent with a two-step reaction:The frst step, with an activation energy EA = 160 kJ/mol, is linked to Schiff base deprotonation; it is endothermic and depends on the hydrophobic milieu around the protein. The second step is slightly exothermic; EA = 60 kJ/mol and n = 2.The transformation of the protein determines the apparent pK, of 6.75. From the known pH dependence of G-protein activation, we conclude that MUI and MUIb must be successively formed to generate full catalytic activity for nucleotide exchange in the G protein.The visual process in rods begins with the absorption of light in rhodopsin (Rh, Am.a = 500 nm), an intrinsic membrane protein in the discs of the rod outer segment. Rh's chromophore retinal forms a protonated Schiffbase with Lys-296, centrally located in the last of the seven transmembrane helices of the apoprotein (1). After rapid cis/trans isomerization of retinal, photoexcited Rh relaxes via intermediates with different chromophore-protein interaction and absorption spectra (2, 3). Metarhodopsin II (MII, A, = 380 nm) forms in milliseconds; its Schiff base is still intact but deprotonated (2, 3). The deprotonation causes the absorption shift to 380 nm (4). It is mandatory for Rh to adopt a conformation in which it interacts with the G protein transducin (4, 5). The MII form remains active for minutes, in equilibrium with the 480-nm intermediate MI (3,6). Kinetics (3,[6][7][8][9] and pH dependence (2, 6) of MII suggest the existence of isochromic MIT species.Besides the protonation change at the Schiff base, light induces the exchange ofprotons between Rh and the aqueous milieu (10). An initial uptake of protons occurs with kineticsThe 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. similar to MIT formation (11), and MIT is favored above MI by acidic pH (2, 3). Schiff base deprotonation and proto...

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Photoactivation of rhodopsin and interaction with transducin in detergent micelles

Cited by 51 publications

References 19 publications

Equilibrium between Metarhodopsin-I and Metarhodopsin-II Is Dependent on the Conformation of the Third Cytoplasmic Loop

Equilibrium between Metarhodopsin-I and Metarhodopsin-II Is Dependent on the Conformation of the Third Cytoplasmic Loop

Modulation of the Metarhodopsin I/Metarhodopsin II Equilibrium of Bovine Rhodopsin by Ionic Strength

Two different forms of metarhodopsin II: Schiff base deprotonation precedes proton uptake and signaling state.

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