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  Voltage Dependence of Proton Pumping by Bacteriorhodopsin Mutants with Altered Lifetime of the M Intermediate

Geibel, S., Lörinczi, E., Bamberg, E., & Friedrich, T. (2013). Voltage Dependence of Proton Pumping by Bacteriorhodopsin Mutants with Altered Lifetime of the M Intermediate. PLoS One, 8(9), e73338-e73338. doi:10.1371/journal.pone.0073338.

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 Creators:
Geibel, Sven1, Author           
Lörinczi, Eva1, Author           
Bamberg, Ernst1, Author           
Friedrich, Thomas1, 2, Author           
Affiliations:
1Department of Biophysical Chemistry, Max Planck Institute of Biophysics, Max Planck Society, ou_2068289              
2Technical University of Berlin, Institute of Chemistry, Berlin, Germany , ou_persistent22              

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 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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Language(s): eng - English
 Dates: 2013-05-312013-07-292013-09-03
 Publication Status: Published online
 Pages: -
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 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1371/journal.pone.0073338
PMID: 24019918
PMC: PMC3760879
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Title: PLoS One
  Abbreviation : PLoS One
Source Genre: Journal
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Publ. Info: San Francisco, CA : Public Library of Science
Pages: - Volume / Issue: 8 (9) Sequence Number: - Start / End Page: e73338 - e73338 Identifier: ISSN: 1932-6203
CoNE: https://pure.mpg.de/cone/journals/resource/1000000000277850