Controlling the pKa of the bacteriorhodopsin Schiff base by use of artificial retinal analogues.

Sheves, Mordechai; Albeck, A.; Friedman, Noga; Ottolenghi, Michael

doi:10.1073/pnas.83.10.3262

Cited by 116 publications

(102 citation statements)

References 27 publications

(34 reference statements)

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“…Reconstitution of Arch3 and four ChR variants with A2 retinal, which carries an extra double bond, resulted in functional pigments with enhanced longwavelength sensitivity 29 . Extensive work on bacteriorhodopsin (BR) [45][46][47][48][49] using synthetic retinal analogues suggests that many other retinal analogues, particularly those with ring modifications, might be applicable for the purpose of chromophore substitution. However, in most cases, it was only shown that the absorption maximum was shifted, without analysing the rhodopsin function.…”

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confidence: 99%

Synthetic retinal analogues modify the spectral and kinetic characteristics of microbial rhodopsin optogenetic tools

et al. 2014

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Optogenetic tools have become indispensable in neuroscience to stimulate or inhibit excitable cells by light. Channelrhodopsin-2 (ChR2) variants have been established by mutating the opsin backbone or by mining related algal genomes. As an alternative strategy, we surveyed synthetic retinal analogues combined with microbial rhodopsins for functional and spectral properties, capitalizing on assays in C. elegans, HEK cells and larval Drosophila. Compared with all-trans retinal (ATR), Dimethylamino-retinal (DMAR) shifts the action spectra maxima of ChR2 variants H134R and H134R/T159C from 480 to 520 nm. Moreover, DMAR decelerates the photocycle of ChR2(H134R) and (H134R/T159C), thereby reducing the light intensity required for persistent channel activation. In hyperpolarizing archaerhodopsin-3 and Mac, naphthyl-retinal and thiophene-retinal support activity alike ATR, yet at altered peak wavelengths. Our experiments enable applications of retinal analogues in colour tuning and altering photocycle characteristics of optogenetic tools, thereby increasing the operational light sensitivity of existing cell lines or transgenic animals.

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confidence: 99%

Synthetic retinal analogues modify the spectral and kinetic characteristics of microbial rhodopsin optogenetic tools

et al. 2014

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“…Thus, a neutral residue at position 85 shifts the pK for SB deprotonation in the dark from -13 (15,16) for wild type to between 7 and 8. These values are close to those reported for SB model compounds in solution (16). The effect of the charge at position 85 on the pK of the SB requires that this residue interacts with and is close to the protonated SB.…”

Section: Discussionmentioning

confidence: 99%

Substitution of amino acids Asp-85, Asp-212, and Arg-82 in bacteriorhodopsin affects the proton release phase of the pump and the pK of the Schiff base.

Otto

Marti

Holz

et al. 1990

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

248

175

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Photocycle and flash-induced proton release and uptake were investigated for bacteriorhodopsin mutants in which Asp-85 was replaced by Ala, Asn, or Glu; Asp-212 was replaced by Asn or Glu; Asp-115 was replaced by Ala, Asn, or Glu; Asp-96 was replaced by Ala, Asn, or Glu; and Arg-82 was replaced by Ala or Gln in dimyristoylphosphatidylcholine/3- [(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate miceiles at pH 7.3. In the Asp-85 -Ala and Asp-85 --Asn mutants, the absence of the charged carboxyl group leads to a blue chromophore at 600 and 595 nm, respectively, and lowers the pK of the Schiff base deprotonation to 8.2 and 7, respectively, suggesting a role for Asp-85 as counterion to the Schiff base. The early part of the photocycles of the Asp-85 -* Ala and Asp-85 -* Asn mutants is strongly perturbed; the formation of a weak M-like intermediate is slowed down about 100-fold over wild type. In both mutants, proton release is also slower but dearly precedes the rise of M. The amplitude of the early (<0.2 ps) reversed photovoltage component in the Asp-85 -* Asn mutant is very large, and the net charge displacement is close to zero, indicating proton release and uptake on the cytoplasmic side of the membrane. The data suggest an obligatory role for Asp-85 in the efficient deprotonation of the Schiff base and in the proton release phase, probably as proton acceptor. In the Asp-212 -* Asn mutant, the rise of the absorbance change at 410 nm is slowed down to 220 Ls, its amplitude is small, and the release of protons is delayed to 1.9 ms. The absorbance changes at 650 nm indicate perturbations in the early time range with a slow K intermediate. Thus Asp-212 also participates in the early events of charge translocation and deprotonation of the Schiff base. In the Arg-82 -Gln mutant, no net transient proton release was observed, whereas, in the Arg-82 Ala mutant,, uptake and release were reversed. The pK shift of the purple-to-blue transition in the Asp-85 --Glu, Arg-82-Ala, and Arg-82 --Gin mutants and the similarity in the photocycle and photoelectrical signals of the Asp-85 -Ala, Asp-85 --Asn, and Asp-212 --Asn mutants suggest the interaction between Asp-85, Arg-82, Asp-212, and the Schiff base as essential for proton release.Site-directed mutagenesis has shown and Asp-212 to be essential for proton translocation by bacteriorhodopsin (bR) (1). The very low activity in mutants Asp-96 -+ Ala (D96A) and Asp-96 -* Asn (D96N) at pH 7 is due to a markedly slowed-down decay of the photocycle intermediate M and the associated charge movement (2-5). was concluded to be the internal proton donor for the reprotonation of the Schiff base (SB) leading to the decay of M (3, 4). Fourier-transform infrared spectroscopy has revealed the protonation states of Asp-85, Asp-96, Asp-115, and Asp-212 in the K, L, and M intermediates and provided clues to the time course of proton transfer (6, 7). The data suggest that Asp-85 and Asp-212 are deprotonated in the ground state and become protonated in the L -* M transition (6). A...

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“…pK a calculations were performed using APBS (58) with the setup described above. All titratable residues were sampled using the Monte Carlo procedure in Karlsberg+, for which a benchmarked accuracy of ∼1 pK a unit has been found (59)(60)(61). VMD (62) was used for analysis and visualization.…”

Section: Methodsmentioning

confidence: 99%

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)...

show abstract

Controlling the pKa of the bacteriorhodopsin Schiff base by use of artificial retinal analogues.

Cited by 116 publications

References 27 publications

Synthetic retinal analogues modify the spectral and kinetic characteristics of microbial rhodopsin optogenetic tools

Synthetic retinal analogues modify the spectral and kinetic characteristics of microbial rhodopsin optogenetic tools

Substitution of amino acids Asp-85, Asp-212, and Arg-82 in bacteriorhodopsin affects the proton release phase of the pump and the pK of the Schiff base.

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

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