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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 MT
.
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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.
Figure 1. Light-induced
change of ATP concentration in halobacterial cells
containing (A) wild-type BR (strain S9) or (B) mutant BR (strain TOM,
two opsin minus, derived from L33 expressing the triple mutant from
a plasmid). Data points shown represent the mean value of three independent
determinations. The standard deviations were less than 10% for all
data points; therefore, the error bars are not shown. For determination
of the initial ATP synthesis rate, the linear regression of the first
five data points was calculated, and the result is indicated by the
dashed lines. (C) Dependence of the ATP synthesis rate on different
light intensities. Note that the y-axis scale in
panels A and B are different.
Figure 2. Quantification of expression
of wild-type and mutant BR in oocytes.
(A) Western blot of total membrane fractions of wild-type (lane 5)
or mutant (lane 6) BR-expressing oocytes. In lanes 1–4, 12.5,
18.8, 25, and 32.2 ng of BR were applied for calibration, respectively.
(B) Densitograms of lanes 1–6. (C) Regression analysis of the
integrals of the curves shown in panel B (standard values, dots; wild-type
BR, open circle; and mutant BR, cross).
Figure 3. Averaged current recordings of oocytes expressing (A) wild-type
BR (n = 18) or (B) mutant BR (n =
19). Voltages are applied in the range of −150 to 50 mV in
20 mV increments from bottom to top. A 1 s light pulse (source: HBO
300, Oriel filtered through OG515, Schott and Calflex 3000, Balzers,
16.3 mW cm–2) was applied between 2.5 and 3.5 s.
The rough illumination region is depicted with a gray rectangle. The
bath solution contained 80 mM NaCl, 3 mM NaN3, 10 mM CsCl,
10 mM TEACl, 5 mM BaCl2, 2 mM CaCl2, 2.5 mM
Na-pyruvate, and 10 mM MES at pH 5.5; electrodes were filled with
3 M KCl.
Figure 4. Averaged current–voltage relationship of wild-type
BR (A)
and mutant BR (B) from 18 and 19 independent measurements, respectively,
as shown in Figure 3. The standard deviation
is indicated by the bars. Experimental conditions were as described
in Figure 3. Note that the y axis in panels A and B is on different scales.
Figure 5. Dynamics in the vicinity of Gly96 in four of the simulations
starting
structure 4FPD. For each simulation, the Gly96–Phe42 side chain Ca–Cg
distance (green), the number of water molecules in the Gly96–Lys216
cavity (red), and the radius of gyration of the leucine-rich cluster
(black) are each plotted against the simulation time. A horizontal
dashed line indicates 4 water molecules, the threshold value for defining
an open channel. See Figure S3 for the
other four simulations.
Figure 6. Representative conformation of the open proton uptake channel.
(A) Model viewed from the CP side. Six water molecules (red balls)
are shown in the Gly96–Lys216 cavity. The opening is surrounded
by a leucine-rich hydrophobic cluster formed by Phe42, Ile45, Leu97,
Leu99, and Leu223 (green stick and yellow surface); (B, C) side view.
The pink pipe depicts a contiguous water channel that connects the
cytoplasm with Lys216 and passes by Gly96.
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