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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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Photocurrents of mutants BR-D96N and BR-D96G.Photocurrents of BR-D96N (A) and BR-D96G (F) induced by illumination with green light (grey bar) at 0 mV (red) and −100 mV (blue). (B,G) Current-voltage plots of normalized stationary photocurrents of BR-D96N (B) and BR-D96G (G) evoked by continuous green light. For each cell, the stationary current amplitude at 0 mV was used for normalization. The dashed lines connecting the data points are drawn to guide the eye; for comparison, the corresponding WT curve from Fig. 2B is included as dotted line. (C,H) Photocurrents of BR-D96N (C, same cell as in panel A) and BR-D96G (H, same cell as in panel F) after addition of 50 mM azide. (D,E,J,K) Green light-induced stationary and blue laser flash-induced transient currents of BR-D96N at 0 mV (D) and −100 mV (E), and of BR-D96G at 0 mV (J) and −100 mV (K). Green light illumination is indicated by grey bars. The shown signals are superpositions of 12 recordings according to the illumination protocol from Fig. 2C. In each sweep, the first blue flash was given at Δt = 100 ms after start and the second at Δt = 100 ms after the end of illumination with green light. From sweep to sweep, Δt increased by 100 ms up to 1200 ms.
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pone-0073338-g004: Photocurrents of mutants BR-D96N and BR-D96G.Photocurrents of BR-D96N (A) and BR-D96G (F) induced by illumination with green light (grey bar) at 0 mV (red) and −100 mV (blue). (B,G) Current-voltage plots of normalized stationary photocurrents of BR-D96N (B) and BR-D96G (G) evoked by continuous green light. For each cell, the stationary current amplitude at 0 mV was used for normalization. The dashed lines connecting the data points are drawn to guide the eye; for comparison, the corresponding WT curve from Fig. 2B is included as dotted line. (C,H) Photocurrents of BR-D96N (C, same cell as in panel A) and BR-D96G (H, same cell as in panel F) after addition of 50 mM azide. (D,E,J,K) Green light-induced stationary and blue laser flash-induced transient currents of BR-D96N at 0 mV (D) and −100 mV (E), and of BR-D96G at 0 mV (J) and −100 mV (K). Green light illumination is indicated by grey bars. The shown signals are superpositions of 12 recordings according to the illumination protocol from Fig. 2C. In each sweep, the first blue flash was given at Δt = 100 ms after start and the second at Δt = 100 ms after the end of illumination with green light. From sweep to sweep, Δt increased by 100 ms up to 1200 ms.

Mentions: Upon expression of wild-type and mutant BR constructs in Xenopus oocytes, transient and stationary photocurrents were measured using the two-electrode voltage-clamp technique. Figure 1 shows the location of the mutated amino acids within the BR structure and illustrates, that all mutated residues are located within the intracellular part (with respect to the retinal) of the proton transport pathway. As observed for BR-WT (Fig. 2A), all investigated mutants respond to green light (λ >495 nm) with positive transient and stationary photocurrents consistent with outwardly directed H+ translocation (Fig. 3A,E; Fig. 4A,F; and Fig. 5A) indicating correct right-side-out insertion in the cellular membrane.


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)

Photocurrents of mutants BR-D96N and BR-D96G.Photocurrents of BR-D96N (A) and BR-D96G (F) induced by illumination with green light (grey bar) at 0 mV (red) and −100 mV (blue). (B,G) Current-voltage plots of normalized stationary photocurrents of BR-D96N (B) and BR-D96G (G) evoked by continuous green light. For each cell, the stationary current amplitude at 0 mV was used for normalization. The dashed lines connecting the data points are drawn to guide the eye; for comparison, the corresponding WT curve from Fig. 2B is included as dotted line. (C,H) Photocurrents of BR-D96N (C, same cell as in panel A) and BR-D96G (H, same cell as in panel F) after addition of 50 mM azide. (D,E,J,K) Green light-induced stationary and blue laser flash-induced transient currents of BR-D96N at 0 mV (D) and −100 mV (E), and of BR-D96G at 0 mV (J) and −100 mV (K). Green light illumination is indicated by grey bars. The shown signals are superpositions of 12 recordings according to the illumination protocol from Fig. 2C. In each sweep, the first blue flash was given at Δt = 100 ms after start and the second at Δt = 100 ms after the end of illumination with green light. From sweep to sweep, Δt increased by 100 ms up to 1200 ms.
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Related In: Results  -  Collection

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pone-0073338-g004: Photocurrents of mutants BR-D96N and BR-D96G.Photocurrents of BR-D96N (A) and BR-D96G (F) induced by illumination with green light (grey bar) at 0 mV (red) and −100 mV (blue). (B,G) Current-voltage plots of normalized stationary photocurrents of BR-D96N (B) and BR-D96G (G) evoked by continuous green light. For each cell, the stationary current amplitude at 0 mV was used for normalization. The dashed lines connecting the data points are drawn to guide the eye; for comparison, the corresponding WT curve from Fig. 2B is included as dotted line. (C,H) Photocurrents of BR-D96N (C, same cell as in panel A) and BR-D96G (H, same cell as in panel F) after addition of 50 mM azide. (D,E,J,K) Green light-induced stationary and blue laser flash-induced transient currents of BR-D96N at 0 mV (D) and −100 mV (E), and of BR-D96G at 0 mV (J) and −100 mV (K). Green light illumination is indicated by grey bars. The shown signals are superpositions of 12 recordings according to the illumination protocol from Fig. 2C. In each sweep, the first blue flash was given at Δt = 100 ms after start and the second at Δt = 100 ms after the end of illumination with green light. From sweep to sweep, Δt increased by 100 ms up to 1200 ms.
Mentions: Upon expression of wild-type and mutant BR constructs in Xenopus oocytes, transient and stationary photocurrents were measured using the two-electrode voltage-clamp technique. Figure 1 shows the location of the mutated amino acids within the BR structure and illustrates, that all mutated residues are located within the intracellular part (with respect to the retinal) of the proton transport pathway. As observed for BR-WT (Fig. 2A), all investigated mutants respond to green light (λ >495 nm) with positive transient and stationary photocurrents consistent with outwardly directed H+ translocation (Fig. 3A,E; Fig. 4A,F; and Fig. 5A) indicating correct right-side-out insertion in the cellular membrane.

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