High-resolution X-ray structure of an early intermediate in the bacteriorhodopsin photocycle

Edman, K. A. P.; Nollert, Peter; Royant, Antoine; Belrhali, Hassan; Pebay‐Peyroula, Eva; Hajdu, János; Neutze, Richard; Landau, Ehud M.

doi:10.1038/44623

Cited by 317 publications

(330 citation statements)

References 30 publications

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“…Cryoelectron microscopy of the K intermediate reported that the structural change at this stage is not resolved with 3.5-Å resolution (21). More recent analysis of the K intermediate by x-ray crystallography identified few changes around the retinal chromophore (22). These results suggest that nuclear motions of the chromophore and surrounding protein would be minimal at the earliest stages.…”

Section: B Acteriorhodopsin (Br) Is a Light-driven Proton Pump Inmentioning

confidence: 83%

Local and distant protein structural changes on photoisomerization of the retinal in bacteriorhodopsin

Kandori

Kinoshita

Yamazaki

et al. 2000

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

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The photoisomerization of the retinal in bacteriorhodopsin is selective and efficient and yields perturbation of the protein structure within femtoseconds. The stored light energy in the primary intermediate is then used for the net translocation of a proton across the membrane in the microsecond to millisecond regime. This study is aimed at identifying how the protein changes on photoisomerization by using the O-H groups of threonines as internal probes. Polarized Fourier-transform IR spectroscopy of [3-18 O]threonine-labeled and unlabeled bacteriorhodopsin indicates that 3 of the threonines (of a total of 18) change their hydrogen bonding. One is exchangeable in D 2O, but two are not. A comprehensive mutation study indicates that the residues involved are Thr-89, Thr-17, and Thr-121 (or Thr-90). The perturbation of only three threonine side chains suggests that the structural alteration at this stage of the photocycle is local and specific. Furthermore, the structural change of Thr-17, which is located >11 Å from the retinal chromophore, implicates a specific perturbation channel in the protein that accompanies the retinal motion. B acteriorhodopsin (BR) is a light-driven proton pump inHalobacterium salinarum that contains all-trans retinal as chromophore (1-4). Its tertiary structure has been determined recently by cryoelectron microscopy (5-7) and x-ray crystallography (8-13). The retinal binds covalently to Lys-216 through a protonated Schiff base linkage. Absorption of light triggers a cyclic reaction that comprises a series of intermediates, designated as the J, K, KL, L, M, N, and O states (1-4). Protein structural changes in these intermediate states cause proton translocation across the protein, and their mechanism is the central question in current studies of BR (14).The all-trans to 13-cis photoisomerization leads to the formation of the primary K intermediate (15)(16)(17). It is well known that photoisomerization in BR is highly selective and efficient. In solution, the photoproduct of all-trans retinal with protonated Schiff base is mainly 11-cis [82% (vol/vol) 11-cis͞6% (vol/vol) 13-cis͞12% (vol/vol) 9-cis in methanol; ref. 18], whereas in BR, the photoproduct is 100% (vol͞vol) 13-cis. The quantum efficiency for isomerization in BR (Ϸ0.6; refs. 19 and 20) is much higher than for retinal in solution (0.13 in methanol; ref. 18). This efficiency is correlated closely with the rate constant of the isomerization, because it occurs on the femtosecond time scale. The protein environment thus certainly facilitates the specificity of the reaction of the retinal chromophore.How does the highly selective and efficient isomerization occur in BR? How does the protein respond to the chromophore motion? To address these questions, structural analysis of the primary intermediates is essential. Cryoelectron microscopy of the K intermediate reported that the structural change at this stage is not resolved with 3.5-Å resolution (21). More recent analysis of the K intermediate by x-ray crystallography identified few...

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Section: B Acteriorhodopsin (Br) Is a Light-driven Proton Pump Inmentioning

confidence: 83%

Local and distant protein structural changes on photoisomerization of the retinal in bacteriorhodopsin

Kandori

Kinoshita

Yamazaki

et al. 2000

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

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“…Crystallographic studies of cryotrapped photocycle intermediates of bacteriorhodopsin have revealed displacements of key water molecules within the proton translocation channel (45)(46)(47). Mechanistic interpretations of such findings must rely on the assumption that the hydration structure is quenched at ambient temperature, rather than at 200 K (as seems more likely).…”

Section: Discussionmentioning

confidence: 99%

Biomolecular cryocrystallography: Structural changes during flash-cooling

Halle

2004

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

188

141

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To minimize radiation damage, crystal structures of biological macromolecules are usually determined after rapid cooling to cryogenic temperatures, some 150 -200 K below the normal physiological range. The biological relevance of such structures relies on the assumption that flash-cooling is sufficiently fast to kinetically trap the macromolecule and associated solvent in a room-temperature equilibrium state. To test this assumption, we use a two-state model to calculate the structural changes expected during rapid cooling of a typical protein crystal. The analysis indicates that many degrees of freedom in a flash-cooled protein crystal are quenched at temperatures near 200 K, where local conformational and association equilibria may be strongly shifted toward low-enthalpy states. Such cryoartifacts should be most important for strongly solvent-coupled processes, such as hydration of nonpolar cavities and surface regions, conformational switching of solventexposed side chains, and weak ligand binding. The dynamic quenching that emerges from the model considered here can also rationalize the glass transition associated with the atomic fluctuations in the protein.protein structure ͉ protein hydration ͉ protein glass transition ͉ x-ray diffraction

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“…The millisecond timescale associated with the long-distance proton transfers may be required for protein conformational changes. The crystal structures of the reaction cycle intermediates indicate rather small structural rearrangements of the protein in K [3,4], L [5][6][7][8][9], and early-M [10]; there are structural rearrangements of the protein during the M-to-N [11][12][13][14][15], and N-to-O transitions [16][17][18]. The passage of the protein through the intermediate conformational states is also accompanied by changes in the number and location of internal water molecules [19,20].…”

Section: Introductionmentioning

confidence: 99%

Mechanism of a proton pump analyzed with computer simulations

2009

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Understanding the mechanism of proton pumping requires a detailed description of the energetics and sequence of events associated with the proton transfers, and of how proton transfer couples to conformational rearrangements of the protein. Here, we discuss our recent advances in using computer simulations to understand how bacteriorhodopsin pumps protons. We emphasize the importance of accurately describing the retinal geometry and the location of water molecules.

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High-resolution X-ray structure of an early intermediate in the bacteriorhodopsin photocycle

Cited by 317 publications

References 30 publications

Local and distant protein structural changes on photoisomerization of the retinal in bacteriorhodopsin

Local and distant protein structural changes on photoisomerization of the retinal in bacteriorhodopsin

Biomolecular cryocrystallography: Structural changes during flash-cooling

Mechanism of a proton pump analyzed with computer simulations

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