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  Stable Closure of the Cytoplasmic Half-Channel Is Required for Efficient Proton Transport at Physiological Membrane Potentials in the Bacteriorhodopsin Catalytic Cycle

Wang, T., Oppawsky, C., Duan, Y., Tittor, J., Oesterhelt, D., & Facciotti, M. T. (2014). Stable Closure of the Cytoplasmic Half-Channel Is Required for Efficient Proton Transport at Physiological Membrane Potentials in the Bacteriorhodopsin Catalytic Cycle. BIOCHEMISTRY, 53(14), 2380-2390. doi:10.1021/bi4013808.

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 Creators:
Wang, Ting1, Author
Oppawsky, Christoph2, Author              
Duan, Yong1, Author
Tittor, Jörg2, Author              
Oesterhelt, Dieter2, Author              
Facciotti, Marc T.1, Author
Affiliations:
1external, ou_persistent22              
2Oesterhelt, Dieter / Membrane Biochemistry, Max Planck Institute of Biochemistry, Max Planck Society, ou_1565164              

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Free keywords: TRANSFORM INFRARED-SPECTROSCOPY; BOUND WATER-MOLECULES; HALOBACTERIUM-HALOBIUM; STRUCTURAL-CHANGES; RELEASE GROUP; M-STATE; ANGSTROM RESOLUTION; VOLTAGE-DEPENDENCE; EARLY INTERMEDIATE; ATP SYNTHESIS
 Abstract: The bacteriorhodopsin (BR) Asp96Gly/Phe171Cys/Phe219Leu triple mutant has been shown to translocate protons 66% as efficiently as the wild-type protein. Light-dependent ATP synthesis in haloarchaeal cells expressing the triple mutant is 85% that of the wild-type BR expressing cells. Therefore, the functional activity of BR seems to be largely preserved in the triple mutant despite the observations that its ground-state structure resembles that of the wild-type M state (i.e., the so-called cytoplasmically open state) and that the mutant shows no significant structural changes during its photocycle, in sharp contrast to what occurs in the wild-type protein in which a large structural opening and closing occurs on the cytoplasmic side. To resolve the contradiction between the apparent functional robustness of the triple mutant and the presumed importance of the opening and dosing that occurs in the wild-type protein, we conducted additional experiments to compare the behavior of wild-type and mutant proteins under different operational loads. Specifically, we characterized the ability of the two proteins to generate light-driven proton currents against a range of membrane potentials. The wild-type protein showed maximal conductance between -150 and -50 mV, whereas the mutant showed maximal conductance at membrane potentials >+50 mV. Molecular dynamics (MD) simulations of the triple mutant were also conducted to characterize structural changes in the protein and in solvent accessibility that might help to functionally contextualize the current-voltage data. These simulations revealed that the cytoplasmic half-channel of the triple mutant is constitutively open and dynamically exchanges water with the bulk. Collectively, the data and simulations help to explain why this mutant BR does not mediate photosynthetic growth of haloarchaeal cells, and they suggest that the structural dosing observed in the wild-type protein likely plays a key role in minimizing substrate back flow in the face of electrochemical driving forces present at physiological membrane potentials.

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Language(s): eng - English
 Dates: 2014
 Publication Status: Published in print
 Pages: 11
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: ISI: 000334657900020
DOI: 10.1021/bi4013808
 Degree: -

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Title: BIOCHEMISTRY
Source Genre: Journal
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Publ. Info: 1155 16TH ST, NW, WASHINGTON, DC 20036 USA : AMER CHEMICAL SOC
Pages: - Volume / Issue: 53 (14) Sequence Number: - Start / End Page: 2380 - 2390 Identifier: ISSN: 0006-2960