title: Stable closure of the cytoplasmic half-channel is required for efficient proton transport at physiological membrane potentials in the bacteriorhodopsin catalytic cycle.
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 closing 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 closing 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.
authors.affiliation: Department of Biomedical Engineering and Genome Center, 451 East Health Science Drive, University of California , Davis, California 95616-8816, United States.
authors.forename: Ting
authors.initials: T
authors.lastname: Wang
authors.forename: Christoph
authors.initials: C
authors.lastname: Oppawsky
authors.forename: Yong
authors.initials: Y
authors.lastname: Duan
authors.forename: Jörg
authors.initials: J
authors.lastname: Tittor
authors.forename: Dieter
authors.initials: D
authors.lastname: Oesterhelt
authors.forename: Marc T
authors.initials: MT
authors.lastname: Facciotti
mesh_terms: D000255:Adenosine Triphosphate / Q000096:biosynthesis / Q000378:metabolism; D000818:Animals; D019843:Archaeal Proteins / Q000378:metabolism*; D001436:Bacteriorhodopsins / Q000378:metabolism*; D055162:Biocatalysis; D003593:Cytoplasm / Q000378:metabolism*; D017136:Ion Transport; D008564:Membrane Potentials; D056004:Molecular Dynamics Simulation; D011522:Protons; D014982:Xenopus laevis
chemical_list: D019843:Archaeal Proteins; D011522:Protons; D001436:Bacteriorhodopsins; D000255:Adenosine Triphosphate