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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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3D structure of BR.Cartoon representation of the 3D structure of bacteriorhodopsin according to the coordinates in PDB structure entry 1C3W by Luecke et al. (1999) prepared with PyMol 1.0 software. The retinal chromophore (magenta) is covalently linked via a Schiff base to Lys-216 in helix G (orange), which - together with the primary proton acceptor (Asp-85) and proton donor group (Asp-96) - is depicted in ball-and-chain representation (oxygen atoms: red, carbon atoms: green, nitrogen atoms: blue). Also shown are Phe-171 and Phe-219, which, together with Asp-96, were mutated herein.
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pone-0073338-g001: 3D structure of BR.Cartoon representation of the 3D structure of bacteriorhodopsin according to the coordinates in PDB structure entry 1C3W by Luecke et al. (1999) prepared with PyMol 1.0 software. The retinal chromophore (magenta) is covalently linked via a Schiff base to Lys-216 in helix G (orange), which - together with the primary proton acceptor (Asp-85) and proton donor group (Asp-96) - is depicted in ball-and-chain representation (oxygen atoms: red, carbon atoms: green, nitrogen atoms: blue). Also shown are Phe-171 and Phe-219, which, together with Asp-96, were mutated herein.

Mentions: The three-dimensional structure of BR-WT reveals seven α-helices (termed A-G) forming a transmembrane pathway for protons, which is subdivided into a cytoplasmic (CP) and an extracellular (EC) part by the chromophore retinal. The retinal is bound via a SB to Lys-216 in helix G (Figure 1). BR mutant structures showed that during the first half of the photocycle (BR→M) only minor structural changes occur, while the relaxation from the state M1 (with SB accessibility still towards EC) through M2 (with SB accessibility towards CP) back to BR is accompanied by distinct, albeit small, structural rearrangements. In detail, after excitation of the retinal, the CP part of the channel still assumes a closed conformation, but deprotonation of the SB and deprotonation of Asp-96 trigger movements of helices B, G and F, which open the CP part of the proton pathway [5], [7], [8], [9], [11], [12]. These studies revealed that well-concerted conformational rearrangements take place during the BR photocycle.


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)

3D structure of BR.Cartoon representation of the 3D structure of bacteriorhodopsin according to the coordinates in PDB structure entry 1C3W by Luecke et al. (1999) prepared with PyMol 1.0 software. The retinal chromophore (magenta) is covalently linked via a Schiff base to Lys-216 in helix G (orange), which - together with the primary proton acceptor (Asp-85) and proton donor group (Asp-96) - is depicted in ball-and-chain representation (oxygen atoms: red, carbon atoms: green, nitrogen atoms: blue). Also shown are Phe-171 and Phe-219, which, together with Asp-96, were mutated herein.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0073338-g001: 3D structure of BR.Cartoon representation of the 3D structure of bacteriorhodopsin according to the coordinates in PDB structure entry 1C3W by Luecke et al. (1999) prepared with PyMol 1.0 software. The retinal chromophore (magenta) is covalently linked via a Schiff base to Lys-216 in helix G (orange), which - together with the primary proton acceptor (Asp-85) and proton donor group (Asp-96) - is depicted in ball-and-chain representation (oxygen atoms: red, carbon atoms: green, nitrogen atoms: blue). Also shown are Phe-171 and Phe-219, which, together with Asp-96, were mutated herein.
Mentions: The three-dimensional structure of BR-WT reveals seven α-helices (termed A-G) forming a transmembrane pathway for protons, which is subdivided into a cytoplasmic (CP) and an extracellular (EC) part by the chromophore retinal. The retinal is bound via a SB to Lys-216 in helix G (Figure 1). BR mutant structures showed that during the first half of the photocycle (BR→M) only minor structural changes occur, while the relaxation from the state M1 (with SB accessibility still towards EC) through M2 (with SB accessibility towards CP) back to BR is accompanied by distinct, albeit small, structural rearrangements. In detail, after excitation of the retinal, the CP part of the channel still assumes a closed conformation, but deprotonation of the SB and deprotonation of Asp-96 trigger movements of helices B, G and F, which open the CP part of the proton pathway [5], [7], [8], [9], [11], [12]. These studies revealed that well-concerted conformational rearrangements take place during the BR photocycle.

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