Replacement of leucine-93 by alanine or threonine slows down the decay of the N and O intermediates in the photocycle of bacteriorhodopsin: implications for proton uptake and 13-cis-retinal----all-trans-retinal reisomerization.

Subramaniam, Sriram; Greenhalgh, David; Rath, Parshuram; Rothschild, Kenneth J.; Khorana, H G

doi:10.1073/pnas.88.15.6873

Cited by 90 publications

(100 citation statements)

References 27 publications

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“…The location of the features observed near helix C in Fig. 2 coincide with the location of Leu-93 in projection, consistent with the suggestion that the contact of Leu-93 with retinal is a key element in mediating conformational changes in the final stages of the photocycle (18,19). In addition to the helix C feature, the maps show evidence for structural changes near helices B, G, and F, which are the three other helices lining the pathway of proton transport (7).…”

Section: Discussionsupporting

confidence: 64%

“…2b) supports this hypothesis. While this difference in the initial conformation of the L93A mutant suggests caution in extending the structural changes observed in the mutant to those that occur in wild-type bacteriorhodopsin, proton pumping experiments show conclusively that the L93A mutant functions as a vectorial proton pump like wild-type bacteriorhodopsin (18). Further, spectroscopic experiments show that the transient absorbance traces observed at different wavelengths can be explained using the same kinetic model and sequence of intermediates used for the wild-type protein (19,23).…”

Section: Discussionmentioning

confidence: 76%

“…Replacement of Leu-93 by Ala (but not by Val) is known to result in a 250-fold decrease in the rate of completion of the photocycle due to the accumulation of a long-lived O intermediate (18). The kinetic defect in the photocycle of the L93A mutant occurs at a stage after the completion of proton release and uptake, and retinal reisomerization is kinetically the rate-limiting step in the photocycle of this mutant (19).…”

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confidence: 99%

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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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Section: Discussionsupporting

confidence: 64%

Section: Discussionmentioning

confidence: 76%

mentioning

confidence: 99%

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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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“…This cavity would be located in a hydrophobic patch as part of the conserved transmembrane helix three (TM3), only a helical turn apart from C128. Manipulating the interaction between C128 (TM3) and D156 (TM4) decelerates the reaction cycle of ChR2 dramatically 5,6 , an effect that was also observed in the bacteriorhodopsin mutant L93A 22,23 , i.e. the neighboring residue of L94.…”

Section: Application To Hippocampal Neuronsmentioning

confidence: 70%

Ultra light-sensitive and fast neuronal activation with the Ca2+-permeable channelrhodopsin CatCh

et al. 2011

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“…The gating function of Leu120 is further supported by the dynamics of the homologous Leu93 in bR, which also shows a flipping behavior (26). Although mutations at Leu120 have not been experimentally studied in KR2 (SI Appendix, Table S2), the photocycle is slowed down by a factor of 100 in the L93A and L93T mutants of bR (27).…”

Section: Significancementioning

confidence: 81%

Energetics and dynamics of a light-driven sodium-pumping rhodopsin

Suomivuori

Gámiz‐Hernández

Sundholm

et al. 2017

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

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The conversion of light energy into ion gradients across biological membranes is one of the most fundamental reactions in primary biological energy transduction. Recently, the structure of the first light-activated Na + pump, Krokinobacter eikastus rhodopsin 2 (KR2), was resolved at atomic resolution [Kato HE, et al. (2015) Nature 521: [48][49][50][51][52][53]. To elucidate its molecular mechanism for Na + pumping, we perform here extensive classical and quantum molecular dynamics (MD) simulations of transient photocycle states. Our simulations show how the dynamics of key residues regulate water and ion access between the bulk and the buried light-triggered retinal site. We identify putative Na + binding sites and show how protonation and conformational changes gate the ion through these sites toward the extracellular side. We further show by correlated ab initio quantum chemical calculations that the obtained putative photocycle intermediates are in close agreement with experimental transient optical spectroscopic data. The combined results of the ion translocation and gating mechanisms in KR2 may provide a basis for the rational design of novel light-driven ion pumps with optogenetic applications.bacterial ion pumps | bioenergetics | QM/MM | optogenetics | retinal P rimary biological energy conversion is based on the efficient capture and conversion of light and chemical energy into ion gradients across biological membranes (1, 2). The established gradients are used to thermodynamically drive energy-requiring processes, such as active transport and synthesis of adenosine triphosphate (ATP) (3, 4). The rhodopsin family of proteins catalyzes such reactions by harnessing the energy from retinal photoisomerization and deprotonation reactions, followed by conformational changes that further trigger the pumping of ions across the membrane (5). All previously known ion-pumping rhodopsins function either as inward chloride or outward proton pumps, but the structure of the first Na + pumping rhodopsin, Krokinobacter eikastus rhodopsin 2 (KR2) (6), was recently resolved (7,8). In the absence of Na + ions, KR2 functions as an outward proton pump, similarly to bacteriorhodopsin (bR), but under physiological conditions, KR2 pumps Na + out of the cell (6, 9).KR2 shares the heptahelical transmembrane (7TM) structure common to all microbial rhodopsins (Fig. 1A) (5,7,8). In contrast to bR, however, where a highly conserved Asp-Thr-Asp (DTD) motif is used to pump protons, KR2 employs a unique NDQ motif comprising residues Asn112, Asp116, and Gln123 (6). In bR, Asp85 and Asp96 function as proton acceptors and donors for the protonated Schiff base (PSB), respectively, whereas in KR2, Asn112 and Gln123 occupy these positions, and Asp116 replaces Thr89. In analogy to bR, it has been suggested that Gln123 aids in capturing ions from the solvent and that Asn112 might be part of a Na + binding site (6,10).Similarly to other microbial rhodopsins, four photocycle intermediates have been spectroscopically identified in KR2 (Fig. 1B) (6)...

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Replacement of leucine-93 by alanine or threonine slows down the decay of the N and O intermediates in the photocycle of bacteriorhodopsin: implications for proton uptake and 13-cis-retinal----all-trans-retinal reisomerization.

Cited by 90 publications

References 27 publications

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

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

Ultra light-sensitive and fast neuronal activation with the Ca2+-permeable channelrhodopsin CatCh

Energetics and dynamics of a light-driven sodium-pumping rhodopsin

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