Bacteriorhodopsin's Intramolecular Proton-Release Pathway Consists of a Hydrogen-Bonded Network

Rammelsberg, Robin; Huhn, Gregor; Lübben, Mathias; Gerwert, Klaus

doi:10.1021/bi971701k

Cited by 203 publications

(273 citation statements)

References 57 publications

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“…Another important consequence of the strong hydrogen bonding interaction is that the COOH band of Glu-194/204 is significantly red-shifted to Ͻ1,700 cm Ϫ1 , falling in the range of amide bands; such strong red-shifts of IR bands are also observed in gas-phase models with a shared proton at both the SCC-DFTB and DFT levels (see SI). This explains why the COOH bands have not been identified in the previous FTIR studies (16,27), which led those authors to pursue alternative models for the PRG.…”

Section: Results and Discussion Structural Features Of Wt Br X-ray Crmentioning

confidence: 99%

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Amino acids with an intermolecular proton bond as proton storage site in bacteriorhodopsin

Phatak

Ghosh

et al. 2008

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

135

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The positions of protons are not available in most high-resolution structural data of biomolecules, thus the identity of proton storage sites in biomolecules that transport proton is generally difficult to determine unambiguously. Using combined quantum mechanical/ molecular mechanical computations, we demonstrate that a pair of conserved glutamate residues (Glu 194/204) bonded by a delocalized proton is the proton release group that has been long sought in the proton pump, bacteriorhodopsin. This model is consistent with all available experimental structural and infrared data for both the wild-type bacteriorhodopsin and several mutants. In particular, the continuum infrared band in the 1,800-to 2,000-cm ؊1 region is shown to arise due to the partially delocalized nature of the proton between the glutamates in the wild-type bacteriorhodopsin; alternations in the flexibility of the glutamates and electrostatic nature of nearby residues in various mutants modulate the degree of proton delocalization and therefore intensity of the continuum band. The strong hydrogen bond between Glu 194/204 also significantly shifts the carboxylate stretches of these residues well <1,700 cm ؊1 , which explains why carboxylate spectral shift was not observed experimentally in the typical >1,700-cm ؊1 region upon proton release. By contrast, simulations with the proton restrained on the nearby water cluster, as proposed by several recent studies [see, for example, Garezarek K, Gerwert K (2006) Functional waters in intraprotein proton transfer monitored by FTIR difference spectroscopy. Nature 439:109], led to significant structural deviations from available X-ray structures. This study establishes a biological function for strong, low-barrier hydrogen bonds.infrared spectroscopy ͉ proton pumping ͉ water cluster ͉ quantum mechanical/molecular mechanical simulations

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Section: Results and Discussion Structural Features Of Wt Br X-ray Crmentioning

confidence: 99%

“…Several mutants have been studied by using the FTIR technique (4,27) to shed light on the detailed structure of the PRG. A critical test for our shared proton model is, therefore, whether it is able to reproduce these findings.…”

Section: Structural and Spectral Features Of Br Mutantsmentioning

confidence: 99%

Amino acids with an intermolecular proton bond as proton storage site in bacteriorhodopsin

Phatak

Ghosh

et al. 2008

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

135

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“…1) (14,22,23). In the ground state, the BR-SB, D85, D212 are all ionized, D96 is neutral, and BR-E194/E204 share a proton or are both ionized with an associated hydronium (22,24). Following isomerization of the retinal from all-trans to 13-cis, the first proton transfer shifts the proton from the SB to D85 (early M state), and then the proton is lost to the periplasm from E194/E204 (late M state).…”

Section: Significancementioning

confidence: 99%

Halorhodopsin pumps Cl^–and bacteriorhodopsin pumps protons by a common mechanism that uses conserved electrostatic interactions

Song¹,

Gunner

2014

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

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Key mutations differentiate the functions of homologous proteins. One example compares the inward ion pump halorhodopsin (HR) and the outward proton pump bacteriorhodopsin (BR). Of the nine essential buried ionizable residues in BR, six are conserved in HR. However, HR changes three BR acids, D85 in a central cluster of ionizable residues, D96, nearer the intracellular, and E204, nearer the extracellular side of the membrane to the small, neutral amino acids T111, V122, and T230, respectively. In BR, acidic amino acids are stationary anions whose proton affinity is modulated by conformational changes, establishing a sequence of directed binding and release of protons. Multiconformation continuum electrostatics calculations of chloride affinity and residue protonation show that, in reaction intermediates where an acid is ionized in BR, a Cl -is bound to HR in a position near the deleted acid. In the HR ground state, Cl -binds tightly to the central cluster T111 site and weakly to the extracellular T230 site, recovering the charges on ionized BR-D85 and neutral E204 in BR. Imposing key conformational changes from the BR M intermediate into the HR structure results in the loss of Cl -from the central T111 site and the tight binding of Cl -to the extracellular T230 site, mirroring the changes that protonate BR-D85 and ionize E204 in BR. The use of a mobile chloride in place of D85 and E204 makes HR more susceptible to the environmental pH and salt concentrations than BR. These studies shed light on how ion transfer mechanisms are controlled through the interplay of protein and ion electrostatics.

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“…These two glutamic acid residues belong to the proton release complex, which releases the proton to the bulk. 60 The temperature factors of S193, E194 and E204 are higher than those of the neighboring residues, indicating that they are more flexible. The higher flexibility of this region makes it likely that the hydroxyl group of S193 is also accessible from the bulk in some conformations.…”

mentioning

confidence: 98%

The Influence of a Transmembrane pH Gradient on Protonation Probabilities of Bacteriorhodopsin: The Structural Basis of the Back-Pressure Effect

Calimet

Ullmann

2004

Journal of Molecular Biology

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Bacteriorhodopsin pumps protons across a membrane using the energy of light. The proton pumping is inhibited when the transmembrane proton gradient that the protein generates becomes larger than four pH units. This phenomenon is known as the back-pressure effect. Here, we investigate the structural basis of this effect by predicting the influence of a transmembrane pH gradient on the titration behavior of bacteriorhodopsin. For this purpose we introduce a method that accounts for a pH gradient in protonation probability calculations. The method considers that in a transmembrane protein, which is exposed to two different aqueous phases, each titratable residue is accessible for protons from one side of the membrane depending on its hydrogen-bond pattern. This method is applied to several ground-state structures of bacteriorhodopsin, which residues already present complicated titration behaviors in the absence of a proton gradient. Our calculations show that a pH gradient across the membrane influences in a non-trivial manner the protonation probabilities of six titratable residues which are known to participate in the proton transfer: D85, D96, D115, E194, E204, and the Schiff base. The residues connected to one side of the membrane are influenced by the pH on the other side because of their long-range electrostatic interactions within the protein. In particular, D115 senses the pH at the cytoplasmic side of the membrane and transmits this information to D85 and the Schiff base. We propose that the strong electrostatic interactions found between D85, D115, and the Schiff base as well as the interplay of their respective protonation states under the influence of a transmembrane pH gradient are responsible for the back-pressure effect on bacteriorhodopsin.

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Bacteriorhodopsin's Intramolecular Proton-Release Pathway Consists of a Hydrogen-Bonded Network

Cited by 203 publications

References 57 publications

Amino acids with an intermolecular proton bond as proton storage site in bacteriorhodopsin

Amino acids with an intermolecular proton bond as proton storage site in bacteriorhodopsin

Halorhodopsin pumps Cl^–and bacteriorhodopsin pumps protons by a common mechanism that uses conserved electrostatic interactions

The Influence of a Transmembrane pH Gradient on Protonation Probabilities of Bacteriorhodopsin: The Structural Basis of the Back-Pressure Effect

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Bacteriorhodopsin's Intramolecular Proton-Release Pathway Consists of a Hydrogen-Bonded Network

Cited by 203 publications

References 57 publications

Amino acids with an intermolecular proton bond as proton storage site in bacteriorhodopsin

Amino acids with an intermolecular proton bond as proton storage site in bacteriorhodopsin

Halorhodopsin pumps Cl–and bacteriorhodopsin pumps protons by a common mechanism that uses conserved electrostatic interactions

The Influence of a Transmembrane pH Gradient on Protonation Probabilities of Bacteriorhodopsin: The Structural Basis of the Back-Pressure Effect

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Halorhodopsin pumps Cl^–and bacteriorhodopsin pumps protons by a common mechanism that uses conserved electrostatic interactions