Chromophore structure in bacteriorhodopsin's O640 photointermediate

Smith, Steven O.; Pardoen, J. A.; Mulder, Patrick P.J.; Curry, Bo; Lugtenburg, J.; Mathies, Richard A.

doi:10.1021/bi00295a016

Cited by 206 publications

(253 citation statements)

References 31 publications

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“…The insensitivity of the v[C( 14)X( 15)] vibration to deuteration supports an anti-configuration of the C = N bond [24]. Similar retinal protein interactions in BRse8, HRsT8 and SRI,, were also concluded from reconstitution experiments with retinal analogs [12,26].…”

Section: Discussionsupporting

confidence: 54%

“…The C(14)-C(15) stretch mode at 1165 cm-' can be used as a marker band for the configuration of the C = N bond due to the different sensitivity to Schiff base deuteration in syn and anti configuration [24]. Deuteration of the SRI,, samples, HR,,, and BR,,, does not lead to a significant up-shift of the mode, which is expected for a syn configuration, indicating an anti geometry in all cases.…”

Section: Spectra Of the Initial Statesmentioning

confidence: 99%

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Sensory rhodopsin I photocycle intermediate SRI₃₈₀ contains 13‐cis retinal bound via an unprotonated Schiff base

et al. 1994

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Sensory rhodopsin I (SRI), the mutated derivative SRI-D76N and the complex of SRI with its transducer HtrI were overexpressed in Halobacterium salinarium and analyzed by resonance Raman spectroscopy. In the initial state SRI contains all-tram retinal bound via a protonated Schiff base as confirmed by retinal extraction which yields 95 f 3% all-trans retinal. The photocycle intermediate absorbing maximally at 380 mn (SRI& contains a Schiff base linkage between the protein and 13-cis retinal. Extraction of illuminated SRI yields up to 93% 13"cis retinal. Neither the mutation D76N nor HtrI changed the vibrational pattern of the chromophore.

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Section: Discussionsupporting

confidence: 54%

Section: Spectra Of the Initial Statesmentioning

confidence: 99%

Sensory rhodopsin I photocycle intermediate SRI₃₈₀ contains 13‐cis retinal bound via an unprotonated Schiff base

et al. 1994

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“…In the wild-type photocycle, retinal is reisomerized to a twisted all-trans configuration (24) It is interesting to speculate on the possible interpretation of the structural changes observed with the L93A mutant, especially in context of the structural work on other mutants. Electron, x-ray, and neutron diffraction experiments with the D96G (or D96N) mutants all are consistent with a conformational change occurring at helix F and G during the photocycle under conditions where the M intermediate is populated.…”

Section: Discussionmentioning

confidence: 99%

Electron diffraction studies of light-induced conformational changes in the Leu-93 → Ala bacteriorhodopsin mutant

Subramaniam

Faruqi

Oesterhelt

et al. 1997

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

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We previously have presented evidence for prominent structural changes in helices F and G of bacteriorhodopsin during the photocycle. These changes were determined by carrying out electron diffraction analysis of illuminated two-dimensional crystals of wild-type bacteriorhodopsin or the Asp-96 3 Gly mutant that were trapped at a stage in the photocycle after light-driven proton release, but preceding proton uptake from the aqueous medium. Here, we report structural analysis of the long-lived O intermediate observed in the photocycle of the Leu-93 3 Ala mutant, which accumulates after the release and uptake of protons, but before the reisomerization of retinal to its initial all-trans state. Projection Fourier difference maps show that upon illumination of the Leu-93 3 Ala mutant, significant structural changes occur in the vicinity of helices C, B, and G, and to a lesser extent near helix F. Our results suggest that (i) all four helices that line the proton channel (B, C, F, and G) participate in structural changes during the late stages of the photocycle, and (ii) completion of the photocycle involves significant conformational changes in addition to those that are associated with steps in proton transport.Bacteriorhodopsin is a light-driven proton pump (1). Each cycle of proton transport is initiated by the all-trans to 13-cis photoisomerization of retinal and completed with the thermal re-isomerization of retinal and return of the protein to its initial conformation. A number of distinct intermediates that occur in the course of the photocycle have been identified by spectroscopic methods (2-4). Site-specific mutagenesis studies (5) and selection of transport-negative mutants (6) have identified key residues that play important roles in the formation and decay of these spectroscopic intermediates. Electron cryomicroscopic studies of two-dimensional crystals have resulted in the determination of a model for the structure of bacteriorhodopsin at atomic resolution (7,8), allowing a structural interpretation of the spectroscopic and biochemical experiments.To understand chemical aspects of the molecular mechanism of proton transport, it is also necessary to determine structural changes in the protein at different stages of the photocycle. A combination of neutron (9), x-ray (10-13), and electron diffraction (14, 15) experiments have begun to provide such information. Two general strategies have been used in these experiments. In one, structural changes have been observed by collecting diffraction data from wild-type bacteriorhodopsin where the photocycle has been slowed down by lowering the temperature, changing the pH, adding chaotropic reagents, or combining two or more of these variables. In the other strategy, mutants that have pronounced kinetic defects in specific stages of the photocycle have been used to trap and structurally characterize the corresponding intermediates. To a first approximation, the magnitude and nature of the structural changes determined by the different methods and by the diff...

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“…Since then, indirect (7) and direct (8) experiments have indicated that the photointermediate K625 also has a 13-cis chromophore. On the other hand, on the basis of Raman spectroscopy evidence, the chromophore in the photointermediate O640 was recently shown to be all-trans (9), as it is in light-adapted bR568 (6). It has been appreciated for many years that C=N bonds can also exhibit geometrical isomerization (10).…”

mentioning

confidence: 99%

Dark-adapted bacteriorhodopsin contains 13-cis, 15-syn and all-trans, 15-anti retinal Schiff bases.

Harbison¹,

Smith²,

Pardoen³

et al. 1984

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

236

157

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ABSTRACT13C NMR spectra of Iyophilized dark-adapted [14-13C]retinyl-labeled bacteriorhodopsin show a large anomalous upfield shift for the 13C-14 resonance assigned to the 13-cis isomer, relative to both the all-trans isomer and model compounds. We attribute this to the so-called y effect, which results from a steric interaction between the C-14 retinal proton and the protons on the e CH2 of the lysine. As a consequence of this observation, we infer that dark-adapted bacteriorhodopsin is composed of a mixture of all-trans,15-anti (trans or E) and 13-cis,15-syn (cis or Z) isomers. These occur in an approximate 4:6 ratio and are commonly identified as bRm and bR54. This conclusion is based on an examination of the isotropic and anisotropic chemical shifts and a comparison with 13C shifts of the carbons adjacent to the C=N linkage in protonated ketinines. Other possible origins for the anomalous shift are examined and shown to be insufficient to account for either the size of the shift or the nature of the shift tensor. We discuss the consequences of this finding for the structure and photochemistry of bacteriorhodopsin.Bacteriorhodopsin (bR), the single protein of the purple membrane (PM) of Halobacterium halobium (1), has been the subject of considerable experimental scrutiny for some time. Like rhodopsin (2), it contains as its chromophore the polyene aldehyde retinal, connected via a Schiff base linkage to the E-amino group of a lysine side chain (3). Although bR appears to resemble visual pigments (4) (14), the configuration of the C=-N Schiff base linkage in bR has not been considered. In addition, the interesting possibility that isomerization about this bond might occur during the photocycle has been largely overlooked. The inattention to this important question can be attributed in part to the absence of an experimental means to discriminate definitively between syn and anti isomers in bR. In a recent paper (15) we demonstrated that high-resolution solid-state 3C NMR is a potent means of establishing configuration about C=C bonds in bR. Here, we present evidence that it is equally effective in determining the C=N bond configuration and evinces that dark-adapted bR contains alltrans,15-anti and 13-cis,15-syn isomers in an approximately 4:6 ratio. The implications of this finding for both the structure of the chromophore and the bR photocycle will also be discussed. MATERIALS AND METHODS13C-14-labeled retinal was prepared by the method of ref. 16 and incorporated into white membrane as described (8, 15). The reconstituted PM was then lyophilized at 0.1 mm Hg (1 mm Hg = 133 Pa) and packed into a Kel-F rotor of the Andrew-Beams design (17). 13C magic-angle sample spinning (MASS) spectra were obtained at various spinning frequencies between 1.9 and 3.2 kHz, with a 13C frequency of 79.9 MHz. Typically, (,)1/21r) = 50 kHz was used for cross-polarization. Subsequently, the magnetization was sampled in the presence of 1H decoupling fields of 125 kHz. Usually, 15,000 transients were accumulated, with a recycle de...

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Chromophore structure in bacteriorhodopsin's O640 photointermediate

Cited by 206 publications

References 31 publications

Sensory rhodopsin I photocycle intermediate SRI₃₈₀ contains 13‐cis retinal bound via an unprotonated Schiff base

Sensory rhodopsin I photocycle intermediate SRI₃₈₀ contains 13‐cis retinal bound via an unprotonated Schiff base

Electron diffraction studies of light-induced conformational changes in the Leu-93 → Ala bacteriorhodopsin mutant

Dark-adapted bacteriorhodopsin contains 13-cis, 15-syn and all-trans, 15-anti retinal Schiff bases.

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Chromophore structure in bacteriorhodopsin's O640 photointermediate

Cited by 206 publications

References 31 publications

Sensory rhodopsin I photocycle intermediate SRI380 contains 13‐cis retinal bound via an unprotonated Schiff base

Sensory rhodopsin I photocycle intermediate SRI380 contains 13‐cis retinal bound via an unprotonated Schiff base

Electron diffraction studies of light-induced conformational changes in the Leu-93 → Ala bacteriorhodopsin mutant

Dark-adapted bacteriorhodopsin contains 13-cis, 15-syn and all-trans, 15-anti retinal Schiff bases.

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Sensory rhodopsin I photocycle intermediate SRI₃₈₀ contains 13‐cis retinal bound via an unprotonated Schiff base

Sensory rhodopsin I photocycle intermediate SRI₃₈₀ contains 13‐cis retinal bound via an unprotonated Schiff base