Specificity of Anion Binding in the Substrate Pocket of Bacteriorhodopsin

Facciotti, Marc T.; Cheung, Vincent S.; Lunde, Christopher S.; Rouhani, Shahab; Baliga, Nitin S.; Glaeser, Robert M.

doi:10.1021/bi035757s

Cited by 14 publications

(11 citation statements)

References 19 publications

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“…1). This assumption is supported by x-ray diffraction results from a similar BR mutant D85S (34,35). The most prominent modification by this mutation is expected to be an altered electrostatic environment around the retinal.…”

Section: Introductionmentioning

confidence: 74%

Influence of the Charge at D85 on the Initial Steps in the Photocycle of Bacteriorhodopsin

Sobotta

Braun

Tittor

et al. 2009

Biophysical Journal

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Studies have shown that trans-cis isomerization of retinal is the primary photoreaction in the photocycle of the light-driven proton pump bacteriorhodopsin (BR) from Halobacterium salinarum, as well as in the photocycle of the chloride pump halorhodopsin (HR). The transmembrane proteins HR and BR show extensive structural similarities, but differ in the electrostatic surroundings of the retinal chromophore near the protonated Schiff base. Point mutation of BR of the negatively charged aspartate D85 to a threonine T (D85T) in combination with variation of the pH value and anion concentration is used to study the ultrafast photoisomerization of BR and HR for well-defined electrostatic surroundings of the retinal chromophore. Variations of the pH value and salt concentration allow a switch in the isomerization dynamics of the BR mutant D85T between BR-like and HR-like behaviors. At low salt concentrations or a high pH value (pH 8), the mutant D85T shows a biexponential initial reaction similar to that of HR. The combination of high salt concentration and a low pH value (pH 6) leads to a subpopulation of 25% of the mutant D85T whose stationary and dynamic absorption properties are similar to those of native BR. In this sample, the combination of low pH and high salt concentration reestablishes the electrostatic surroundings originally present in native BR, but only a minor fraction of the D85T molecules have the charge located exactly at the position required for the BR-like fast isomerization reaction. The results suggest that the electrostatics in the native BR protein is optimized by evolution. The accurate location of the fixed charge at the aspartate D85 near the Schiff base in BR is essential for the high efficiency of the primary reaction.

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Section: Introductionmentioning

confidence: 74%

Influence of the Charge at D85 on the Initial Steps in the Photocycle of Bacteriorhodopsin

Sobotta

Braun

Tittor

et al. 2009

Biophysical Journal

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“…However, this situation differs from the anion coordination in the BR mutant D85S. Here, D212 has to be present in the deprotonated state to coordinate the anion (26,27) together with the protonated Schiff base and the hydroxyl of S85 and a water molecule. No coordination was reported with T89 which is also highly conserved among different BRs.…”

Section: Discussionmentioning

confidence: 99%

The Crystal Structure of the L1 Intermediate of Halorhodopsin at 1.9 Å Resolution†

Gmelin

Zeth

Efremov³

et al. 2007

Photochemistry and Photobiology

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The mutant T203V of the light driven chloride pump halorhodopsin from Halobacterium salinarum was crystallized and the X-ray structure was solved at 1.6 angstroms resolution. The T203V structure turned out to be nearly identical to the wild type protein with a root mean square deviation of 0.43 angstroms for the carbon alpha atoms of the protein backbone. Two chloride binding (CB) sites were demonstrated by a substitution of chloride with bromide and an analysis of anomalous difference Fourier maps. The CB1 site was found at the same position as in the wild type structure. In addition, a second chloride binding site CB2 was identified around Q105 due to higher resolution in the mutant crystal. As T203V showed a 10 times slower decay of its photocycle intermediate L, this intermediate could be trapped with an occupancy of 60% upon illumination at room temperature and subsequent cooling to 120 degrees K. Fourier transform infrared spectroscopy clearly identified the crystal to be trapped in the L1 intermediate state and the X-ray structure was solved to 1.9 angstroms resolution. In this intermediate, the chloride moved by 0.3 angstroms within binding site CB1 as indicated by peaks in difference Fourier density maps. The chloride in the second binding site CB2 remained unchanged. Thus, intraproteinous chloride translocation from the extracellular to the cytoplasmic part of the protein must occur in reaction steps following the L1 intermediate in the catalytic cycle of halorhodopsin.

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“…9 Since the protonation state of Asp85 remains unchanged through the N and O intermediates, 41,42 it seems plausible that the conformation of the proton release channel found in M RT Glaeser and his colleagues have recently reported the structures of the D85S mutant of bacteirorhodopsin, which is able to accommodate an anion (chloride, bromide or nitrate) in the vicinity of the Schiff base. 20,43,44 In the presence of halide ions, this mutant exhibits purple. It has been shown that a halide ion binds to the active site and interacts directly with the protonated Schiff base.…”

Section: Crystal Structures Of Bacteriorhodopsinmentioning

confidence: 99%