Time-Resolved FTIR Spectroscopy of the Photointermediates Involved in Fast Transient H<sup>+</sup> Release by Proteorhodopsin

Xiao, Yaowu; Partha, Ranga; Krebs, R.; Braiman, Mark S.

doi:10.1021/jp046314g

Cited by 27 publications

(53 citation statements)

References 42 publications

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“…1). The green-absorbing PR is activated at alkaline pH in either a phospholipid or detergent environment, 13,16,17 which corresponds well with the pH of the ocean near the surface (~8.0). Photoexcitation of PR results in the translocation of a proton across the membrane.…”

Section: Introductionmentioning

confidence: 71%

“…The proteorhodopsin photocycle when the protein is suspended in a (A) phospholipid bilayer, 14,16,19 or (B) detergent suspension. 9,11 Lifetimes are shown for select photostates that have been resolved in the cited references.…”

Section: Figurementioning

confidence: 99%

“…3–7 These proteins are identified solely from genetic analysis of coastal and oceanic water samples and no native host has yet been cultivated. 4,8 Hence, PRs are often expressed in Escherichia coli and solubilized in various detergents 9–12 or reconstituted in phospholipid environments 13–16 to stabilize the protein prior to investigation.…”

Section: Introductionmentioning

confidence: 99%

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Green Proteorhodopsin Reconstituted into Nanoscale Phospholipid Bilayers (Nanodiscs) as Photoactive Monomers

et al. 2011

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Over 4,000 putative proteorhodopsins (PRs) have been identified throughout the oceans and seas of the Earth. The first of these eubacterial rhodopsins was discovered in 2000 and has expanded the family of microbial proton pumps to all three domains of life. With photophysical properties similar to those of bacteriorhodopsin, an archaeal proton pump, PRs are also generating interest for their potential use in various photonic applications. We perform here the first reconstitution of the minimal photoactive PR structure into nanoscale phospholipid bilayers (nanodiscs), to better understand how protein-protein and protein-lipid interactions influence the photophysical properties of PR. Spectral (steady-state and time-resolved UV-visible spectroscopy) and physical (size-exclusion chromatography and electron microscopy) characterization of these complexes confirms the preparation of a photoactive PR monomer within nanodiscs. Specifically, when embedded within a nanodisc, monomeric PR exhibits a titratable pKa (6.5–7.1) and photocycle lifetime (~100–200 ms) that is comparable to the detergent solubilized protein. These ndPRs also produce a photoactive blue-shifted absorbance, centered at 377 or 416 nm, that indicates protein-protein interactions from a PR oligomer are required for a fast photocycle. Moreover, we demonstrate how these model membrane systems allow modulation of the PR photocycle by variation of the discoidal diameter (i.e., 10 or 12 nm), bilayer thickness (i.e., 23 or 26.5 Å), and degree of saturation of the lipid acyl chain. Nanodiscs also offer a highly stable environment of relevance to potential device applications.

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Section: Introductionmentioning

confidence: 71%

Section: Figurementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Green Proteorhodopsin Reconstituted into Nanoscale Phospholipid Bilayers (Nanodiscs) as Photoactive Monomers

et al. 2011

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“…On the other hand, measurements by Krebs et al (53) at pH 9.5 in diheptanoylphosphatidylcholine micelles resulted in a proton signal of reversed polarity: proton release first (20 Ws), followed by proton uptake (0.3 s) somewhat earlier than the decay of the slow component of M and completion of the photocycle (0.6 s) occur (53). Subsequent FTIR study suggested that the release cannot be from Asp97 since it remained protonated in this time domain (54). A study of the pH dependence of proton release and uptake with a pH sensitive SnO 2 electrode by Tamogami et al confirmed these two different kinds of signals (55), at neutral and high pH.…”

Section: Discussionmentioning

confidence: 99%

Aspartate–Histidine Interaction in the Retinal Schiff Base Counterion of the Light-Driven Proton Pump of Exiguobacterium sibiricum

et al. 2012

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One of the distinctive features of eubacterial retinal based proton pumps, proteorhodopsins, xanthorhodopsin and others, is hydrogen bonding of the key aspartate residue, the counterion to the retinal Schiff base, to a histidine. We describe properties of the recently found eubacterium proton pump from Exiguobacterium sibiricum (named ESR) expressed in E. coli, especially features that depend on Asp-His interaction, the protonation state of the key aspartate, Asp85, and its ability to accept proton from the Schiff base during the photocycle. Proton pumping by liposomes and E. coli cells containing ESR occurs in a broad pH range above pH 4.5. Large light-induced pH changes indicate that ESR is a potent proton pump. Replacement of His57 with methionine or asparagine strongly affects the pH dependent properties of ESR. In the H57M mutant a dramatic decrease in the quantum yield of chromophore fluorescence emission and a 45 nm blue shift of the absorption maximum upon raising the pH from 5 to 8 indicates deprotonation of the counterion with a pKa of 6.3, which is also the pKa at which the M intermediate is observed in the photocycle of the protein solubilized in detergent (DDM). This is in contrast with the wild type protein, in which the same experiments show that the major fraction of Asp85 is deprotonated at pH > 3 and that it protonates only at low pH, with a pKa of 2.3. The M intermediate in the wild type photocycle accumulates only at high pH, with an apparent pKa of 9 from deprotonation of a residue interacting with Asp85, presumably His57. In liposomes reconstituted with ESR the pKas for M formation and spectral shifts are 2–3 pH units lower than in DDM. The distinctively different pH dependencies of the protonation of Asp85 and the accumulation of the M intermediate in the wild type protein vs. the H57M mutant indicate that there is strong Asp-His interaction, which substantially lowers the pKa of Asp85 by stabilizing its deprotonated state.

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“…A similar band in the BR photocycle was assigned to a Glu side chain acting as proton donor. In analogy the corresponding band in PR is tentatively assigned to a partial deprotonation or a disturbance of Glu108, although this is still under discussion (18). This band is a marker for the L‐intermediate at low pH.…”

mentioning

confidence: 99%

Characterizing the Structure and Photocycle of PR 2D Crystals with CD and FTIR Spectroscopy^†

Schäfer

Shastri

Verhoefen

et al. 2009

Photochem & Photobiology

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We present here a study on proteorhodopsin (PR) 2D crystals with analytical ultracentrifugation, circular dichroism and Fourier transform infrared (FTIR) spectroscopy. The aim of our experiments was to test the activity of 2D crystal sample preparations and to gain further insight in PR structure, stability and function with these techniques. Our results demonstrate higher stability compared to detergent-solubilized or reconstituted samples. For different pH values, low pH 2D crystals tend to form bigger aggregates and are less stable than at basic pH. The pH 9 sample shows a sharp phase transition during heat denaturation and there is also evidence for protein-protein interaction due to the close proximity of the proteins in the 2D crystals. In the FTIR measurements at cryogenic temperatures (77 K), we characterized the first step in the PR photocycle. At pH 9, the K intermediate could be observed and the samples showed no orientation effects. At pH 5, we could trap the K/L intermediate, characterized by its negative IR signal at 1741 cm(-1). In rapid-scan FTIR experiments, we could also identify the M intermediate of the photocycle at basic pH. We conclude that the PR 2D crystals exhibit a fully functional photocycle and are therefore well suited for further studies on the proton transport mechanism of PR.

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Time-Resolved FTIR Spectroscopy of the Photointermediates Involved in Fast Transient H⁺ Release by Proteorhodopsin

Cited by 27 publications

References 42 publications

Green Proteorhodopsin Reconstituted into Nanoscale Phospholipid Bilayers (Nanodiscs) as Photoactive Monomers

Green Proteorhodopsin Reconstituted into Nanoscale Phospholipid Bilayers (Nanodiscs) as Photoactive Monomers

Aspartate–Histidine Interaction in the Retinal Schiff Base Counterion of the Light-Driven Proton Pump of Exiguobacterium sibiricum

Characterizing the Structure and Photocycle of PR 2D Crystals with CD and FTIR Spectroscopy^†

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Time-Resolved FTIR Spectroscopy of the Photointermediates Involved in Fast Transient H+ Release by Proteorhodopsin

Cited by 27 publications

References 42 publications

Green Proteorhodopsin Reconstituted into Nanoscale Phospholipid Bilayers (Nanodiscs) as Photoactive Monomers

Green Proteorhodopsin Reconstituted into Nanoscale Phospholipid Bilayers (Nanodiscs) as Photoactive Monomers

Aspartate–Histidine Interaction in the Retinal Schiff Base Counterion of the Light-Driven Proton Pump of Exiguobacterium sibiricum

Characterizing the Structure and Photocycle of PR 2D Crystals with CD and FTIR Spectroscopy†

Contact Info

Product

Resources

About

Time-Resolved FTIR Spectroscopy of the Photointermediates Involved in Fast Transient H⁺ Release by Proteorhodopsin

Characterizing the Structure and Photocycle of PR 2D Crystals with CD and FTIR Spectroscopy^†