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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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Comparison of characteristic properties of photocurrent signals.Photocurrents in response to continuous green light are characterized by five parameters: the amplitude of the transient current peak at the beginning of illumination (a), a stationary current amplitude (b), an initial amplitude of the slow phase of current decay after the end of illumination (c), a relaxation time τ1 for the decrease from the initial peak current to the stationary level and τ3 for the slow current decay after the end of illumination. The time constants for the initial current increase (τ0) at the beginning and for the initial current decrease after light switch-off (τ2) are not resolved due to the limited time resolution in TEVC experiments. In each panel, the parameters for the shown signals are included for comparison.
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pone-0073338-g006: Comparison of characteristic properties of photocurrent signals.Photocurrents in response to continuous green light are characterized by five parameters: the amplitude of the transient current peak at the beginning of illumination (a), a stationary current amplitude (b), an initial amplitude of the slow phase of current decay after the end of illumination (c), a relaxation time τ1 for the decrease from the initial peak current to the stationary level and τ3 for the slow current decay after the end of illumination. The time constants for the initial current increase (τ0) at the beginning and for the initial current decrease after light switch-off (τ2) are not resolved due to the limited time resolution in TEVC experiments. In each panel, the parameters for the shown signals are included for comparison.

Mentions: The transient current preceding the stationary current reflecting the reaction BR→M [1], is similar to BR-WT. The stationary current amplitude is determined by the reactions M1→M2→BR, as long as BR→M is much faster. Changes in the kinetics of these reactions would strongly influence the Ipeak/Istationary ratio. However, the Ipeak/Istationary value of BR-F171C is comparable to that of BR-WT indicating a similarly fast photocycle. The decay phase (off-response) of mutant BR-F171C exhibits two exponential components with time constants similar to BR-WT (see Fig. 6) indicating that the rate-limiting step of the transport cycle is not significantly altered. These time constants agree well with the time constants of 3 ms and 8 ms determined at pH 7 by time-resolved FT-IR spectroscopy for the de- and reprotonation of Asp-96, respectively [27], [28]. In notable contrast to BR-WT, the fast component has a larger amplitude implying that the major fraction of BR-F171C molecules, which decay to the ground state in the dark reaction does not generate large charge movement. This observation on the N-accumulating BR-F171C mutant is in line with the interpretation that the electrogenicity of the N→O→BR partial reaction sequence is smaller than that of the preceding M→N transition. Blue laser flashes applied to BR-F171C during green illumination did not result in negative transient currents at 0 mV (Fig. 3C), and at −100 mV negative transients were only small (Fig. 3D) indicating that the accumulation of M intermediates during illumination is drastically reduced. This property of BR-F171C (indicating that SB reprotonation proceeds faster to bring about M decay in favor of N) is remarkable considering that Phe-171 resides at the largest distance to the SB from all amino acids studied herein.


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)

Comparison of characteristic properties of photocurrent signals.Photocurrents in response to continuous green light are characterized by five parameters: the amplitude of the transient current peak at the beginning of illumination (a), a stationary current amplitude (b), an initial amplitude of the slow phase of current decay after the end of illumination (c), a relaxation time τ1 for the decrease from the initial peak current to the stationary level and τ3 for the slow current decay after the end of illumination. The time constants for the initial current increase (τ0) at the beginning and for the initial current decrease after light switch-off (τ2) are not resolved due to the limited time resolution in TEVC experiments. In each panel, the parameters for the shown signals are included for comparison.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0073338-g006: Comparison of characteristic properties of photocurrent signals.Photocurrents in response to continuous green light are characterized by five parameters: the amplitude of the transient current peak at the beginning of illumination (a), a stationary current amplitude (b), an initial amplitude of the slow phase of current decay after the end of illumination (c), a relaxation time τ1 for the decrease from the initial peak current to the stationary level and τ3 for the slow current decay after the end of illumination. The time constants for the initial current increase (τ0) at the beginning and for the initial current decrease after light switch-off (τ2) are not resolved due to the limited time resolution in TEVC experiments. In each panel, the parameters for the shown signals are included for comparison.
Mentions: The transient current preceding the stationary current reflecting the reaction BR→M [1], is similar to BR-WT. The stationary current amplitude is determined by the reactions M1→M2→BR, as long as BR→M is much faster. Changes in the kinetics of these reactions would strongly influence the Ipeak/Istationary ratio. However, the Ipeak/Istationary value of BR-F171C is comparable to that of BR-WT indicating a similarly fast photocycle. The decay phase (off-response) of mutant BR-F171C exhibits two exponential components with time constants similar to BR-WT (see Fig. 6) indicating that the rate-limiting step of the transport cycle is not significantly altered. These time constants agree well with the time constants of 3 ms and 8 ms determined at pH 7 by time-resolved FT-IR spectroscopy for the de- and reprotonation of Asp-96, respectively [27], [28]. In notable contrast to BR-WT, the fast component has a larger amplitude implying that the major fraction of BR-F171C molecules, which decay to the ground state in the dark reaction does not generate large charge movement. This observation on the N-accumulating BR-F171C mutant is in line with the interpretation that the electrogenicity of the N→O→BR partial reaction sequence is smaller than that of the preceding M→N transition. Blue laser flashes applied to BR-F171C during green illumination did not result in negative transient currents at 0 mV (Fig. 3C), and at −100 mV negative transients were only small (Fig. 3D) indicating that the accumulation of M intermediates during illumination is drastically reduced. This property of BR-F171C (indicating that SB reprotonation proceeds faster to bring about M decay in favor of N) is remarkable considering that Phe-171 resides at the largest distance to the SB from all amino acids studied herein.

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