Two-photon spectroscopy of locked-11-cis-rhodopsin: evidence for a protonated Schiff base in a neutral protein binding site.

Birge, Robert R.; Murray, Lionel P.; Pierce, Brian M.; Akita, H.; Balogh‐Nair, Valeria; Findsen, Leonore A.; Nakanishi, Koji

doi:10.1073/pnas.82.12.4117

Cited by 133 publications

(121 citation statements)

References 28 publications

(61 reference statements)

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“…3A) (30)(31)(32)(33). As previously reported (13), in Rh, the 34% of the chromophore charge, originally located on the -NHACH-moiety, is shifted toward the ␤-ionone upon S 0 3 S 1 vertical excitation.…”

Section: Resultssupporting

confidence: 64%

The color of rhodopsins at theab initiomulticonfigurational perturbation theory resolution

Coto

Strambi

Ferré

et al. 2006

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

131

204

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We demonstrate that ''brute force'' quantum-mechanics͞molecu-lar-mechanics computations based on ab initio (i.e., first principles) multiconfigurational perturbation theory can reproduce the absorption maxima of a set of modified bovine rhodopsins with an accuracy allowing for the analysis of the factors determining their colors. In particular, we show that the theory accounts for the changes in excitation energy even when the proteins display the same charge distribution. Three color-tuning mechanisms, leading to changes of close magnitude, are demonstrated to operate in these conditions. The first is based on the change of the conformation of the conjugated backbone of the retinal chromophore. The second operates through the control of the distance between the positive charge residing on the chromophore and the carboxylate counterion. Finally, the third mechanism operates through the changes in orientation of the chromophore relative to the protein. These results offer perspectives for the unbiased computational design of mutants or chemically modified proteins with wanted optical properties.excited states ͉ quantum mechanics͞molecular mechanics R ecently, protein mutants displaying properties such as high binding affinities and novel catalytic activities (1, 2) have been designed by using computational methods based on molecular mechanics. In a close context, substrate selectivity has been simulated by using more advanced tools based on density functional theory and molecular mechanics paving the way to the design of mutations that convert receptors into enzymes (3). However, the design of mutants with specific spectral properties, such as color and luminescence, represents a more complex problem. In these cases, the quantum chemical method used must be capable to describe both ground and electronically excited states of the protein chromophore. The ab initio (i.e., first-principles) complete-active-space self-consistent-field (CASSCF) method (4) is a multiconfigurational method offering maximum flexibility for an unbiased description of the electronic and equilibrium structure of a molecule (i.e., with no empirically derived parameters and avoiding single-reference wavefunctions). Furthermore, the CASSCF wave function can be used for subsequent multiconfigurational second-order perturbation theory (5) computations (CASPT2) of the dynamic correlation energy of each state ultimately leading to a nearly quantitative evaluation of the excitation energies of organic compounds (6, 7). The CASPT2 ability to describe exotic bonding has been recently assessed (8).In a previous study (9), we implemented the ab initio CASPT2͞͞CASSCF protocol (where equilibrium geometries and electronic energies are determined at the CASSCF and CASPT2 levels, respectively) in a quantum mechanics͞ molecular mechanics (QM͞MM) scheme (10-12) allowing for the evaluation of the excitation energy of chromophores (treated quantum mechanically) embedded in a protein environment (described by a MM force field).The absorption and fluorescence max...

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Section: Resultssupporting

confidence: 64%

The color of rhodopsins at theab initiomulticonfigurational perturbation theory resolution

Coto

Strambi

Ferré

et al. 2006

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

131

204

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“…The results show that specific portions of TM helices 3 and 6 in rhodopsin comprise specific retinal-protein contacts that define the chromophore-binding pocket. The present results are consistent with a model of rhodopsin based on data from electron microscopy studies (6), NMR and two-photon spectroscopy measurements (7)(8)(9), and a comparative analysis of G protein-coupled receptors (10). A molecular graphics model of the transmembrane domain of rhodopsin is presented that illustrates the relative proximity of the retinal chromophore to Gly 121 and Phe 261 .…”

supporting

confidence: 86%

Functional Interaction of Transmembrane Helices 3 and 6 in Rhodopsin

Han

Lin

Minkova

et al. 1996

Journal of Biological Chemistry

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Replacement of a highly conserved glycine residue on transmembrane (TM) helix 3 of bovine rhodopsin (Gly 121 ) by amino acid residues with larger side chains causes a progressive blue-shift in the max value of the pigment, a decrease in thermal stability, and an increase in reactivity with hydroxylamine. In addition, mutation of Gly 121 causes a relative reversal in the selectivity of opsin for 11-cis-retinal over all-trans-retinal. We show that the loss of function phenotypes of the G121L mutant described above can be partially reverted specifically by the mutation of Phe 261 , a residue highly conserved in all G protein-coupled receptors. For example, the double-replacement mutant G121L/F261A has spectral, chromophore-binding, and transducin-activating properties intermediate between those of G121L and rhodopsin. This rescue of the G121L defects did not occur with the other second site mutations tested. We conclude that specific portions of TM helices 3 and 6, which include Gly 121 and Phe 261 , respectively, define the chromophore-binding pocket in rhodopsin. Finally, the results are placed in the context of a molecular graphics model of the TM domain of rhodopsin, which includes the retinal-binding pocket.G protein-coupled receptors, including the visual pigment rhodopsin, have been studied extensively by site-directed mutagenesis. In many cases, the biochemical phenotype of a mutant receptor involves the loss of a particular function. Unfortunately, a loss of function can be related either directly or indirectly to a particular amino acid replacement. It is often difficult to distinguish between direct and indirect effects. As a result, such studies often do not produce significant structural or mechanistic insights. Certain strategies have evolved to circumvent the problem of interpreting loss of function phenotypes. These include the construction of chimeric receptors (1, 2), the use of biophysical methods to assay defective mutants (3, 4), and the use of genetic selection methods, when available.In the preceding paper (5), we presented a study of a highly conserved glycine residue on transmembrane (TM) 1 helix 3 of bovine rhodopsin (Gly 121 . The results show that specific portions of TM helices 3 and 6 in rhodopsin comprise specific retinal-protein contacts that define the chromophore-binding pocket. The present results are consistent with a model of rhodopsin based on data from electron microscopy studies (6), NMR and two-photon spectroscopy measurements (7-9), and a comparative analysis of G protein-coupled receptors (10). A molecular graphics model of the transmembrane domain of rhodopsin is presented that illustrates the relative proximity of the retinal chromophore to Gly 121 and Phe 261 . Since Phe 261 is highly conserved among all G protein-coupled receptors (11), these results may be relevant to understanding the molecular mechanisms of activation of G protein-coupled receptors.EXPERIMENTAL PROCEDURES TM helix 6 mutant genes were generated by substituting a 45-base pair MluI-NdeI restriction ...

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“…The effects of other charged amino acids and of aromatic amino acids on the opsin shift must be evaluated as well. If other charged amino acid side chains cannot be demonstrated to interact with the chromophore, then the chromophore binding site may be neutral as proposed previously (24,28).…”

Section: Methodsmentioning

confidence: 97%

Glutamic acid-113 serves as the retinylidene Schiff base counterion in bovine rhodopsin.

Sakmar

Franke

Khorana

1989

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

685

626

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The characteristic wavelength at which a visual pigment absorbs light is regulated by interactions between protein (opsin) and retinylidene Schiff base chromophore. By using site-directed mutagenesis, charged amino acids in bovine rhodopsin transmembrane helix C were systematically replaced. Substitution of glutamic acid-134 or arginine-135 did not affect spectral properties. However, substitution of glutamic acid-122 by glutamine or by aspartic acid formed pigments that were blue-shifted in light absorption (Amax = 480 nm and 475 nm, respectively). While the substitution of glutamic acid-113 by aspartic acid gave a slightly red-shifted pigment (Amax = 505 nm), replacement by glutamine formed a pigment that was strikingly blue-shifted in light absorption (Amax = 380 nm). The 380-nm species existed in a pH-dependent equilibrium with a 490-nm species such that at acidic pH all of the pigment was converted to Amax = 490 nm. We conclude that glutamic acid-113 serves as the retinylidene Schiff base counterion in rhodopsin. We believe that this opsin-chromophore interaction is an example of a general mechanism of color regulation in the visual pigments.

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Two-photon spectroscopy of locked-11-cis-rhodopsin: evidence for a protonated Schiff base in a neutral protein binding site.

Cited by 133 publications

References 28 publications

The color of rhodopsins at theab initiomulticonfigurational perturbation theory resolution

The color of rhodopsins at theab initiomulticonfigurational perturbation theory resolution

Functional Interaction of Transmembrane Helices 3 and 6 in Rhodopsin

Glutamic acid-113 serves as the retinylidene Schiff base counterion in bovine rhodopsin.

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