Water Structural Changes at the Proton Uptake Site (the Thr46-Asp96 Domain) in the L Intermediate of Bacteriorhodopsin

Yamazaki, Yoichi; Hatanaka, Minoru; Kandori, Hideki; Sasaki, Jun; Karstens, Willem F. J.; Raap, J.; Lugtenburg, J.; Bizounok, Marina; Herzfeld, Judith

doi:10.1021/bi00021a021

Cited by 65 publications

(108 citation statements)

References 32 publications

(43 reference statements)

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“…These residues are located near D96, and the OH group of T46 is hydrogen-bonded to the carboxyl group of D96 (28,29). These results suggest that perturbation of the environment of D96 stabilizes the hydrogen bond between D96 and neighboring water molecules (30) and prevents the recovery of high pK of D96, which is needed for reprotonation of D96 in the photocycle. If the structural transition from M-type to N-type has the role of introducing water molecule(s) into the proton channel and lowering the D96 pK value, proton transfer from D96 to the Schiff base would occur after this structural transition.…”

Section: Discussionmentioning

confidence: 89%

Time-resolved x-ray diffraction reveals multiple conformations in the M–N transition of the bacteriorhodopsin photocycle

Oka

Yagi

Fujisawa

et al. 2000

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

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We measured the M-N transition of wild-type bacteriorhodopsin (pH 9, 10°C) by time-resolved x-ray diffraction study at SPring8 BL45XU-A. We confirmed the accumulation of M and N intermediates by absorbance measurements, and we found that the time resolution of x-ray diffraction experiments (244 ms) was sufficient to resolve the M-N transition. From the x-ray diffraction data, three components were decomposed by singular value decomposition analysis. The existence of three components in the M3 N3 BR reaction revealed that BR changes its structure during the M-N transition. Moreover, the difference Fourier maps of reconstituted fast and slow decay components clearly showed that the electron density distributions of the F helix changes in the M-N transition. The observed structural change at the F helix will increase access of the Schiff base and D96 to the cytoplasmic surface and facilitate the proton transfer steps that begin with the decay of the M state.A n ion pump protein transports ions across the membrane against the chemical potential. A simple idea of the ion pumping mechanism is that the ion-binding states of the ion pump are linked to protein conformations to switch the ion transport pathway from one membrane surface to another surface (1). Bacteriorhodopsin (BR) is one of the proton transport proteins that make use of absorbed photon energy. Its photocycle is characterized by distinct spectra of the metastable intermediate in the visible range as J, K, L, M, N, and O. The most important steps for the proton pump are deprotonation and reprotonation of the Schiff base. The former is the proton transfer from the Schiff base to D85 and the latter is the proton transfer from D96 to the Schiff base. Proton transfer from the Schiff base to D85 occurs in the L-M transition. The structure of the M intermediate was investigated by diffraction methods under stabilizing conditions (2-4). The obtained data showed that the structure of the M intermediate differed largely from the unphotolyzed state. It was shown that this structural change is closely related to the deprotonation of the Schiff base; in the original structure (conformation E) the proton channel is open to the extracellular side, and when an M-type conformation is assumed (conformation C), it is open to the cytoplasmic side (5-7). This structural transition after the first proton transfer prevents reverse proton transfer from D85 to the Schiff base. Thus, the alternating protein conformation model explains the proton transport mechanism (7). The Schiff base is reprotonated from D96 during the M-N transition. Because D96 is located in a hydrophobic environment, it has unusual high pK and it is protonated in an unphotolyzed state. The pK of D96 should be lowered during the M-N transition to transfer a proton. Therefore, an important event in lowering the D96 pK is expected to occur at the M-N transition.Structural studies on photointermediates have been carried out by accumulating specific intermediates using chemical treatments (3, 4), mutations (8, 9),...

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Section: Discussionmentioning

confidence: 89%

Time-resolved x-ray diffraction reveals multiple conformations in the M–N transition of the bacteriorhodopsin photocycle

Oka

Yagi

Fujisawa

et al. 2000

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

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“…After reaction of 4 with thionyl chloride, the freshly prepared acetyl chloride was reacted with diazomethane to afford a 75% yield of [1,2-13 C 2 ]3-benzyloxy-1-diazopropanone (5). Heating of 5 with dibenzyl phosphate in toluene gave [2,[3][4][5][6][7][8][9][10][11][12][13] C 2 ]dibenzyl-3-benzyloxypropanone phosphate (6) in 93% yield, which could subsequently be deprotected with Pd/C and H 2 to yield [2,3-13 C 2 ]dihydroxyacetone monophosphate (7).…”

Section: Resultsmentioning

confidence: 99%

“…solid-state NMR, [3] EPR, [4] FTIR, [5] and UV Laser Resonance Raman [6] spectroscopy) provides valuable information concerning the structure and function of the isotopically enriched site at an atomic level. In our research programme, this strategy has been successfully used to probe light-driven processes in the membrane proteins bacteriorhodopsin (a 26 kDa proton pump) and bovine rhodopsin (a 40 kDa visual pigment), as well as those occurring at photosynthetic reaction centres (100 kDa).…”

Section: Introductionmentioning

confidence: 99%

Chemo-Enzymatic Synthesis of Thymidine13C-Labelled in the 2′-Deoxyribose Moiety

Ouwerkerk¹,

Boom²,

Lugtenburg³

et al. 2000

Eur. J. Org. Chem.

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A synthesis of [3′,4′‐13C2]thymidine (1) is described in which [13C2]acetic acid (2) is converted into the nucleoside in twelve steps with 9% overall yield. D‐2‐Deoxyribose‐5‐phosphate aldolase (DERA, EC 4.1.2.4) and triosephosphate isomerase (TPI, EC 5.3.1.1) are used for the stereocontrolled formation of D‐[3,4‐13C2]‐2‐deoxyribose‐5‐phosphate (8) from [2,3‐13C2]dihydroxyacetone monophosphate (DHAP, 7) and acetaldehyde in 80% yield. The route permits the introduction of isotopically enriched carbon atoms at any position or combination of positions in the furanose ring and the product can be coupled with any of the four naturally occurring base moieties.

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“…A chain of ionic and hydrogen bonds consisting of protein residues and bound water is believed to mediate long-range interactions between the Schiff base and the cytoplasmically accessible residues participating in proton pumping by bacteriorhodopsin a I I I (26)(27)(28). Because HtrI-free SRI translocates protons (25), it is reasonable to suggest this aspect of bacteriorhodopsin structure is conserved.…”

Section: Methodsmentioning

confidence: 99%

Protonatable residues at the cytoplasmic end of transmembrane helix-2 in the signal transducer HtrI control photochemistry and function of sensory rhodopsin I.

Jung

Spudich

1996

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

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Neutral residue replacements were made of 21 acidic and basic residues within the N-terminal half of the Halobacterium salinarium signal transducer HtrI [the halobacterial transducer for sensory rhodopsin I (SRI)] by site-specific mutagenesis. The replacements are all within the region of HtrI that we previously concluded from deletion analysis to contain sites of interaction with the phototaxis receptor SRI. Immunoblotting shows plasmid expression of the htrI-sopI operon containing the mutations produces SRI and mutant HtrI in cells at near wild-type levels. Six of the HtrI mutations perturb photochemical kinetics of SRI and one reverses the phototaxis response. Substitution with neutral amino acids of Asp-86, Glu-87, and Glu-108 accelerate, and of Arg-70, Arg-84, and Arg-99 retard, the SRI photocycle. Opposite effects on photocycle rate cancel in double mutants containing one replaced acidic and one replaced basic residue. Laser flash spectroscopy shows the kinetic perturbations are due to alteration of the rate of reprotonation of the retinylidene Schiff base. All of these mutations permit normal attractant and repellent signaling. On the other hand, the substitution of Glu-56 with the isosteric glutamine converts the normally attractant effect of orange light to a repellent signal in vivo at neutral pH (inverted signaling). Low pH corrects the inversion due to Glu-56 -> Gln and the apparent pK of the inversion is increased when arginine is substituted at position 56. The results indicate that the cytoplasmic end of transmembrane helix-2 and the initial part of the cytoplasmic domain contain interaction sites with SRI. To explain these and previous results, we propose a model in which (i) the HtrI region identified here forms part of an electrostatic bonding network that extends through the SRI protein and includes its photoactive site; (ii) alteration of this network by photoisomerization-induced Schiff base deprotonation and reprotonation shifts HtrI between attractant and repellent conformations; and (iii) HtrI mutations and extracellular pH alter the equilibrium ratios of these conformations.Photon absorption by the seven-transmembrane helix phototaxis receptor sensory rhodopsin I (SRI; Ama 587 nm) in Halobacterium salinarium membranes causes transient deprotonation of the retinylidene Schiff base and formation of the spectrally distinct species S373 (Amax 373 nm) (1). This process acts as an attractant signal for the cell, and absorption of a second photon by S373 acts as a repellent signal (2, 3). SRI phototransformations are detected by an integral membrane protein, HtrI, a 57-kDa methyl-accepting transducer (4) that exhibits sequence similarity to eubacterial chemotaxis receptors (5) and is complexed with SRI (6, 7). The sequencepredicted structure of HtrI consists of 2 transmembrane helices within the N terminal "60 residues followed by an '500-residue cytoplasmic domain. The C terminal 250 residues contain signaling regions that regulate a cytoplasmic pathway (8) which controls flagellar mot...

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Water Structural Changes at the Proton Uptake Site (the Thr46-Asp96 Domain) in the L Intermediate of Bacteriorhodopsin

Cited by 65 publications

References 32 publications

Time-resolved x-ray diffraction reveals multiple conformations in the M–N transition of the bacteriorhodopsin photocycle

Time-resolved x-ray diffraction reveals multiple conformations in the M–N transition of the bacteriorhodopsin photocycle

Chemo-Enzymatic Synthesis of Thymidine13C-Labelled in the 2′-Deoxyribose Moiety

Protonatable residues at the cytoplasmic end of transmembrane helix-2 in the signal transducer HtrI control photochemistry and function of sensory rhodopsin I.

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