Christoph Oppawsky

Max Planck Institute of Biochemistry, München, Bavaria, Germany

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Publications (4)4.68 Total impact

  • [Show abstract] [Hide abstract]
    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. The functional activity of BR seems to be therefore largely preserved in the triple mutant despite the observation 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 to -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 functionally contextualize the current-voltage data. These simulations revealed the cytoplasmic half-channel of the triple mutant is constitutively "open" and dynamically exchanges water with the bulk. Collectively the data and simulations help explain why this mutant BR does not mediate photosynthetic growth of haloarchaeal cells and 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.
    Biochemistry 03/2014; · 3.38 Impact Factor
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    ABSTRACT: The archaeon Halobacterium salinarum can grow phototrophically with only light as its energy source. It uses the retinal containing and light-driven proton pump bacteriorhodopsin to enhance the membrane potential which drives the ATP synthase. Therefore, a model of the membrane potential generation of bacteriorhodopsin is of central importance to the development of a mathematical model of the bioenergetics of H. salinarum. To measure the current produced by bacteriorhodopsin at different light intensities and clamped voltages, we expressed the gene in Xenopus laevis oocytes. We present current-voltage measurements and a mathematical model of the current-voltage relationship of bacteriorhodopsin and its generation of the membrane potential. The model consists of three intermediate states, the BR, L, and M states, and comparisons between model predictions and experimental data show that the L to M reaction must be inhibited by the membrane potential. The model is not able to fit the current-voltage measurements when only the M to BR phase is membrane potential dependent, while it is able to do so when either only the L to M reaction or both reactions (L to M and M to BR) are membrane potential dependent. We also show that a decay term is necessary for modeling the rate of change of the membrane potential.
    Mathematical biosciences 02/2010; 225(1):68-80. · 1.30 Impact Factor
  • Mathematical Biosciences, v.225, 68-80 (2010).
  • Christoph Oppawsky