Cooperation among c-subunits of FoF1-ATP synthase in rotation-coupled proton translocation
- Faculty of Education, Tokoha University, Japan
- Department of Chemistry and Biochemistry, National Institute of Technology, Numazu College, Japan
- Department of Chemical and Biological Engineering, National Institute of Technology, Ube College, Japan
- Department of Biophysics, Graduate School of Science, Kyoto University, Japan
Figures
Figure 1
Schematic picture of the a-subunit and c-ring of Fo.
(a) The ac10 part of the Fo region is depicted as a ribbon diagram. Spheres represent cE59, which was substituted in this study, aE223, aE162, and aR176 (blue) (the residue numbers are those from …
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Figure 1—source data 1
Schematic picture of the a-subunit and c-ring of Fo.
- https://cdn.elifesciences.org/articles/69096/elife-69096-fig1-data1-v1.pdf
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Figure 1—source data 2
Schematic diagram of simulation model.
- https://cdn.elifesciences.org/articles/69096/elife-69096-fig1-data2-v1.pdf
Figure 2
Expression of the mutated Fo-c subunit and proton pump, and ATP synthesis activities of membrane vesicles containing mutated FoF1s.
(a) Proteins were separated using SDS-PAGE and immunoblotted with anti-Fo-c antibodies. (b) ATP synthesis driven by NADH oxidation. The rightmost bars [E56Q] show the results of c10(E56Q)-FoF1. …
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Figure 2—source data 1
Expression of the mutated Fo-c subunit.
- https://cdn.elifesciences.org/articles/69096/elife-69096-fig2-data1-v1.zip
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Figure 2—source data 2
ATP synthesis activities of membrane vesicles containing mutated FoF1s.
- https://cdn.elifesciences.org/articles/69096/elife-69096-fig2-data2-v1.xlsx
Figure 3 with 2 supplements
Proton transfer-coupled MD simulation of the WT and hetero mutants with Asp substitution of Glu.
(a) Ten trajectories of the “e” mutant. The black line shows one representative trajectory. Upper part: rotation angle from initial position of c(a); lower part: the number of protons that entered …
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Figure 3—source data 1
Proton transfer-coupled MD simulation of the WT and hetero mutants with Asp substitution of Glu.
- https://cdn.elifesciences.org/articles/69096/elife-69096-fig3-data1-v1.zip
Figure 3—figure supplement 1
(a) Simulation overview; (b) visualized cE59 (left) and cE59D (right); (c) the transfer efficiency under the effect of mutation.
The plot is exp(−A(r−r0)). (A, r0)=(2.5/nm, 0.8 nm) for cE59 (black plot), and (A, r0)=(9.0/nm, 0.6 nm) for cE59D (red plot).
Figure 3—figure supplement 2
The average rotation velocities of WT and mutant c-rings in MD simulations with different parameter sets in the cE59D mutant.
(a) A=2.5/nm, pKa=7.0 for cE59D. The decay rate, A, was set to the same value as in the WT. (b) A=9.0/nm, pKa=7.8 for cE59D. The pKa value of cE59D was set to be 0.2 units smaller than that of cE59. …
Figure 4
Analysis of the molecular simulations.
(a) Schematic graph of duration times. Total time was divided into the duration for proton release, the duration for deprotonated rotation, the duration for proton uptake, and the resting time. (b) …
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Figure 4—source data 1
Analysis of the molecular simulations.
- https://cdn.elifesciences.org/articles/69096/elife-69096-fig4-data1-v1.zip
Tables
Table 1
P-value of ATP synthesis activity between the two mutants.
| e | ef | eg | eh | ei | ej | |
|---|---|---|---|---|---|---|
| c10 | 5.41×10–7 | 1.08×10–8 | 6.44×10–9 | 2.45×10–9 | 1.88×10–9 | 1.73×10–8 |
| e | 5.29×10–3 | 9.14×10–4 | 3.54×10–5 | 1.61×10–5 | 6.25×10–6 | |
| ef | 0.409 | 0.0357 | 0.0435 | 0.0122 | ||
| eg | 0.179 | 0.241 | 0.0752 | |||
| eh | 0.706 | 0.784 | ||||
| ei | 0.420 |
Table 2
Membrane ATPase activity from cells expressing hetero-mutated c-subunits.
| ATPase activity* | ||
|---|---|---|
| Mutant | –DCCD | +DCCD |
| WT | 0.23 | 0.076 |
| c10-fusion | 0.15 | 0.067 |
| Mutant e | 0.087 | 0.076 |
| Mutant ef | 0.090 | 0.065 |
| Mutant eg | 0.078 | 0.070 |
| Mutant eh | 0.086 | 0.073 |
| Mutant ei | 0.088 | 0.080 |
| Mutant ej | 0.083 | 0.068 |
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*
Membrane ATPase activity was measured after pre-incubation of membranes at 10 mg/ml in PA3 buffer with or without 50 μM DCCD for 20 min at 25°C. Activity is expressed as μmol/min/mg.
Table 3
pKa values predicted by PROPKA.
| c-ring | a | b | c | d | e | f | g | h | i | j |
|---|---|---|---|---|---|---|---|---|---|---|
| cE59 | 7.40 | 6.10 | 5.89 | 5.89 | 5.89 | 5.89 | 5.89 | 6.73 | 8.03 | 7.34 |
| cE59D | 6.49 | 5.37 | 5.16 | 5.16 | 5.16 | 5.16 | 5.16 | 5.85 | 7.08 | 6.54 |
