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Voltage dependence of proton pumping by bacteriorhodopsin mutants with altered lifetime of the M intermediate.

Geibel S, Lörinczi É, Bamberg E, Friedrich T - PLoS ONE (2013)

Bottom Line: Hyperpolarizing potentials augmented these effects.However, BR-tri showed negative blue laser flash-induced currents even without actinic green light, indicating that Schiff base deprotonation in BR-tri exists in the dark, in line with previous spectroscopic investigations.Thus, M-stabilizing mutations, including the triple mutation, drastically interfere with electrochemical H(+) gradient generation.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institute of Biophysics, Department of Biophysical Chemistry, Frankfurt am Main, Germany.

ABSTRACT
The light-driven proton pump bacteriorhodopsin (BR) from Halobacterium salinarum is tightly regulated by the [H(+)] gradient and transmembrane potential. BR exhibits optoelectric properties, since spectral changes during the photocycle are kinetically controlled by voltage, which predestines BR for optical storage or processing devices. BR mutants with prolonged lifetime of the blue-shifted M intermediate would be advantageous, but the optoelectric properties of such mutants are still elusive. Using expression in Xenopus oocytes and two-electrode voltage-clamping, we analyzed photocurrents of BR mutants with kinetically destabilized (F171C, F219L) or stabilized (D96N, D96G) M intermediate in response to green light (to probe H(+) pumping) and blue laser flashes (to probe accumulation/decay of M). These mutants have divergent M lifetimes. As for BR-WT, this strictly correlates with the voltage dependence of H(+) pumping. BR-F171C and BR-F219L showed photocurrents similar to BR-WT. Yet, BR-F171C showed a weaker voltage dependence of proton pumping. For both mutants, blue laser flashes applied during and after green-light illumination showed reduced M accumulation and shorter M lifetime. In contrast, BR-D96G and BR-D96N exhibited small photocurrents, with nonlinear current-voltage curves, which increased strongly in the presence of azide. Blue laser flashes showed heavy M accumulation and prolonged M lifetime, which accounts for the strongly reduced H(+) pumping rate. Hyperpolarizing potentials augmented these effects. The combination of M-stabilizing and -destabilizing mutations in BR-D96G/F171C/F219L (BR-tri) shows that disruption of the primary proton donor Asp-96 is fatal for BR as a proton pump. Mechanistically, M destabilizing mutations cannot compensate for the disruption of Asp-96. Accordingly, BR-tri and BR-D96G photocurrents were similar. However, BR-tri showed negative blue laser flash-induced currents even without actinic green light, indicating that Schiff base deprotonation in BR-tri exists in the dark, in line with previous spectroscopic investigations. Thus, M-stabilizing mutations, including the triple mutation, drastically interfere with electrochemical H(+) gradient generation.

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Structural details during the BR photocycle.Three-dimensional ball and stick representations of the retinal chromophore (yellow), coupled via a Schiff base to Lys-216 (nitrogen atom in red) are shown and distances from the Schiff base nitrogen to the carboxyl oxygens (black) of Asp-85 are indicated by green dashed lines according to the following structural coordinates: (A) BR ground state structure (PDB structure entry 1C3W) [9], (B) BR ground state structure (PDB structure entry 1FBB) [41], (C) structure of the M intermediate (PDB structure entry 1C3W) [9], (D) Ground state structure of the triple mutant BR-D96G/F171C/F219L (PDB structure entry 1FBK) [11].
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pone-0073338-g007: Structural details during the BR photocycle.Three-dimensional ball and stick representations of the retinal chromophore (yellow), coupled via a Schiff base to Lys-216 (nitrogen atom in red) are shown and distances from the Schiff base nitrogen to the carboxyl oxygens (black) of Asp-85 are indicated by green dashed lines according to the following structural coordinates: (A) BR ground state structure (PDB structure entry 1C3W) [9], (B) BR ground state structure (PDB structure entry 1FBB) [41], (C) structure of the M intermediate (PDB structure entry 1C3W) [9], (D) Ground state structure of the triple mutant BR-D96G/F171C/F219L (PDB structure entry 1FBK) [11].

Mentions: Some features of the triple mutant, however, differ markedly from all other mutants. Firstly, the transient current (on-response) has a different shape compared to BR-WT. Rising and falling phase are slower, which can be interpreted as a slower initial phase of the photocycle (usually the formation of M). This is in line with the slower M formation, as determined by spectroscopy (1 ms, vs. 50 µs for BR-WT) in a study of Tittor and colleagues [14]. How can this behavior be explained? In Figure 7, the distances between the SB and the carboxyl oxygens of Asp-85 are depicted according to structural information for BR-WT and BR-tri. The structures provided by Sass et al. for BR-WT (ground state and M) showed that the distance increases during the BR→M transition (Fig. 7A,C). The BR-tri structure by Subramaniam et al. [11] revealed that (1) the distance between Asp-85 and the SB is increased in the unilluminated ground state already, and (2) that the CP channel is opened (Fig. 7B,D). The increased distance between Asp-85 and the SB may consequently slow down SB deprotonation towards Asp-85 and the formation of M in BR-tri. Furthermore, the primary proton donor for SB reprotonation is removed due to the D96G mutation, which most likely also entails a slowed-down SB reprotonation during the photocycle of BR-tri. Both properties, in effect, would make BR-tri a slow and inefficient proton pump.


Voltage dependence of proton pumping by bacteriorhodopsin mutants with altered lifetime of the M intermediate.

Geibel S, Lörinczi É, Bamberg E, Friedrich T - PLoS ONE (2013)

Structural details during the BR photocycle.Three-dimensional ball and stick representations of the retinal chromophore (yellow), coupled via a Schiff base to Lys-216 (nitrogen atom in red) are shown and distances from the Schiff base nitrogen to the carboxyl oxygens (black) of Asp-85 are indicated by green dashed lines according to the following structural coordinates: (A) BR ground state structure (PDB structure entry 1C3W) [9], (B) BR ground state structure (PDB structure entry 1FBB) [41], (C) structure of the M intermediate (PDB structure entry 1C3W) [9], (D) Ground state structure of the triple mutant BR-D96G/F171C/F219L (PDB structure entry 1FBK) [11].
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3760879&req=5

pone-0073338-g007: Structural details during the BR photocycle.Three-dimensional ball and stick representations of the retinal chromophore (yellow), coupled via a Schiff base to Lys-216 (nitrogen atom in red) are shown and distances from the Schiff base nitrogen to the carboxyl oxygens (black) of Asp-85 are indicated by green dashed lines according to the following structural coordinates: (A) BR ground state structure (PDB structure entry 1C3W) [9], (B) BR ground state structure (PDB structure entry 1FBB) [41], (C) structure of the M intermediate (PDB structure entry 1C3W) [9], (D) Ground state structure of the triple mutant BR-D96G/F171C/F219L (PDB structure entry 1FBK) [11].
Mentions: Some features of the triple mutant, however, differ markedly from all other mutants. Firstly, the transient current (on-response) has a different shape compared to BR-WT. Rising and falling phase are slower, which can be interpreted as a slower initial phase of the photocycle (usually the formation of M). This is in line with the slower M formation, as determined by spectroscopy (1 ms, vs. 50 µs for BR-WT) in a study of Tittor and colleagues [14]. How can this behavior be explained? In Figure 7, the distances between the SB and the carboxyl oxygens of Asp-85 are depicted according to structural information for BR-WT and BR-tri. The structures provided by Sass et al. for BR-WT (ground state and M) showed that the distance increases during the BR→M transition (Fig. 7A,C). The BR-tri structure by Subramaniam et al. [11] revealed that (1) the distance between Asp-85 and the SB is increased in the unilluminated ground state already, and (2) that the CP channel is opened (Fig. 7B,D). The increased distance between Asp-85 and the SB may consequently slow down SB deprotonation towards Asp-85 and the formation of M in BR-tri. Furthermore, the primary proton donor for SB reprotonation is removed due to the D96G mutation, which most likely also entails a slowed-down SB reprotonation during the photocycle of BR-tri. Both properties, in effect, would make BR-tri a slow and inefficient proton pump.

Bottom Line: Hyperpolarizing potentials augmented these effects.However, BR-tri showed negative blue laser flash-induced currents even without actinic green light, indicating that Schiff base deprotonation in BR-tri exists in the dark, in line with previous spectroscopic investigations.Thus, M-stabilizing mutations, including the triple mutation, drastically interfere with electrochemical H(+) gradient generation.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institute of Biophysics, Department of Biophysical Chemistry, Frankfurt am Main, Germany.

ABSTRACT
The light-driven proton pump bacteriorhodopsin (BR) from Halobacterium salinarum is tightly regulated by the [H(+)] gradient and transmembrane potential. BR exhibits optoelectric properties, since spectral changes during the photocycle are kinetically controlled by voltage, which predestines BR for optical storage or processing devices. BR mutants with prolonged lifetime of the blue-shifted M intermediate would be advantageous, but the optoelectric properties of such mutants are still elusive. Using expression in Xenopus oocytes and two-electrode voltage-clamping, we analyzed photocurrents of BR mutants with kinetically destabilized (F171C, F219L) or stabilized (D96N, D96G) M intermediate in response to green light (to probe H(+) pumping) and blue laser flashes (to probe accumulation/decay of M). These mutants have divergent M lifetimes. As for BR-WT, this strictly correlates with the voltage dependence of H(+) pumping. BR-F171C and BR-F219L showed photocurrents similar to BR-WT. Yet, BR-F171C showed a weaker voltage dependence of proton pumping. For both mutants, blue laser flashes applied during and after green-light illumination showed reduced M accumulation and shorter M lifetime. In contrast, BR-D96G and BR-D96N exhibited small photocurrents, with nonlinear current-voltage curves, which increased strongly in the presence of azide. Blue laser flashes showed heavy M accumulation and prolonged M lifetime, which accounts for the strongly reduced H(+) pumping rate. Hyperpolarizing potentials augmented these effects. The combination of M-stabilizing and -destabilizing mutations in BR-D96G/F171C/F219L (BR-tri) shows that disruption of the primary proton donor Asp-96 is fatal for BR as a proton pump. Mechanistically, M destabilizing mutations cannot compensate for the disruption of Asp-96. Accordingly, BR-tri and BR-D96G photocurrents were similar. However, BR-tri showed negative blue laser flash-induced currents even without actinic green light, indicating that Schiff base deprotonation in BR-tri exists in the dark, in line with previous spectroscopic investigations. Thus, M-stabilizing mutations, including the triple mutation, drastically interfere with electrochemical H(+) gradient generation.

Show MeSH
Related in: MedlinePlus