Structural Asymmetry and Kinetic Limping of Single Rotary F-ATP Synthases
F-ATP synthases use proton flow through the F<sub>O</sub> domain to synthesize ATP in the F<sub>1</sub> domain. In <i>Escherichia coli</i>, the enzyme consists of rotor subunits γε<b>c</b><sub>10</sub> and stator subunits...
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MDPI AG
2019-01-01
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| Series: | Molecules |
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| Online Access: | https://www.mdpi.com/1420-3049/24/3/504 |
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| author | Hendrik Sielaff Seiga Yanagisawa Wayne D. Frasch Wolfgang Junge Michael Börsch |
| author_facet | Hendrik Sielaff Seiga Yanagisawa Wayne D. Frasch Wolfgang Junge Michael Börsch |
| author_sort | Hendrik Sielaff |
| collection | DOAJ |
| description | F-ATP synthases use proton flow through the F<sub>O</sub> domain to synthesize ATP in the F<sub>1</sub> domain. In <i>Escherichia coli</i>, the enzyme consists of rotor subunits γε<b>c</b><sub>10</sub> and stator subunits (αβ)<sub>3</sub>δ<b>ab</b><sub>2</sub>. Subunits <b>c</b><sub>10</sub> or (αβ)<sub>3</sub> alone are rotationally symmetric. However, symmetry is broken by the <b>b</b><sub>2</sub> homodimer, which together with subunit δ<b>a</b>, forms a single eccentric stalk connecting the membrane embedded F<sub>O</sub> domain with the soluble F<sub>1</sub> domain, and the central rotating and curved stalk composed of subunit γε. Although each of the three catalytic binding sites in (αβ)<sub>3</sub> catalyzes the same set of partial reactions in the time average, they might not be fully equivalent at any moment, because the structural symmetry is broken by contact with <b>b</b><sub>2</sub>δ in F<sub>1</sub> and with <b>b</b><sub>2</sub><b>a</b> in F<sub>O</sub>. We monitored the enzyme’s rotary progression during ATP hydrolysis by three single-molecule techniques: fluorescence video-microscopy with attached actin filaments, Förster resonance energy transfer between pairs of fluorescence probes, and a polarization assay using gold nanorods. We found that one dwell in the three-stepped rotary progression lasting longer than the other two by a factor of up to 1.6. This effect of the structural asymmetry is small due to the internal elastic coupling. |
| first_indexed | 2024-04-13T16:09:23Z |
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| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-04-13T16:09:23Z |
| publishDate | 2019-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Molecules |
| spelling | doaj.art-ef8643f45cef41e886da790a759eb0f02022-12-22T02:40:18ZengMDPI AGMolecules1420-30492019-01-0124350410.3390/molecules24030504molecules24030504Structural Asymmetry and Kinetic Limping of Single Rotary F-ATP SynthasesHendrik Sielaff0Seiga Yanagisawa1Wayne D. Frasch2Wolfgang Junge3Michael Börsch4Single-Molecule Microscopy Group, Jena University Hospital, Friedrich Schiller University, 07743 Jena, GermanySchool of Life Sciences, Arizona State University, Tempe, Arizona, AZ 85287, USASchool of Life Sciences, Arizona State University, Tempe, Arizona, AZ 85287, USADepartment of Biology & Chemistry, University of Osnabrück, 49076 Osnabrück, GermanySingle-Molecule Microscopy Group, Jena University Hospital, Friedrich Schiller University, 07743 Jena, GermanyF-ATP synthases use proton flow through the F<sub>O</sub> domain to synthesize ATP in the F<sub>1</sub> domain. In <i>Escherichia coli</i>, the enzyme consists of rotor subunits γε<b>c</b><sub>10</sub> and stator subunits (αβ)<sub>3</sub>δ<b>ab</b><sub>2</sub>. Subunits <b>c</b><sub>10</sub> or (αβ)<sub>3</sub> alone are rotationally symmetric. However, symmetry is broken by the <b>b</b><sub>2</sub> homodimer, which together with subunit δ<b>a</b>, forms a single eccentric stalk connecting the membrane embedded F<sub>O</sub> domain with the soluble F<sub>1</sub> domain, and the central rotating and curved stalk composed of subunit γε. Although each of the three catalytic binding sites in (αβ)<sub>3</sub> catalyzes the same set of partial reactions in the time average, they might not be fully equivalent at any moment, because the structural symmetry is broken by contact with <b>b</b><sub>2</sub>δ in F<sub>1</sub> and with <b>b</b><sub>2</sub><b>a</b> in F<sub>O</sub>. We monitored the enzyme’s rotary progression during ATP hydrolysis by three single-molecule techniques: fluorescence video-microscopy with attached actin filaments, Förster resonance energy transfer between pairs of fluorescence probes, and a polarization assay using gold nanorods. We found that one dwell in the three-stepped rotary progression lasting longer than the other two by a factor of up to 1.6. This effect of the structural asymmetry is small due to the internal elastic coupling.https://www.mdpi.com/1420-3049/24/3/504F<sub>O</sub>F<sub>1</sub> ATP synthase<i>Escherichia coli</i>single-molecule fluorescencesymmetrycryo-EM structuresubunit rotationelasticity |
| spellingShingle | Hendrik Sielaff Seiga Yanagisawa Wayne D. Frasch Wolfgang Junge Michael Börsch Structural Asymmetry and Kinetic Limping of Single Rotary F-ATP Synthases Molecules F<sub>O</sub>F<sub>1</sub> ATP synthase <i>Escherichia coli</i> single-molecule fluorescence symmetry cryo-EM structure subunit rotation elasticity |
| title | Structural Asymmetry and Kinetic Limping of Single Rotary F-ATP Synthases |
| title_full | Structural Asymmetry and Kinetic Limping of Single Rotary F-ATP Synthases |
| title_fullStr | Structural Asymmetry and Kinetic Limping of Single Rotary F-ATP Synthases |
| title_full_unstemmed | Structural Asymmetry and Kinetic Limping of Single Rotary F-ATP Synthases |
| title_short | Structural Asymmetry and Kinetic Limping of Single Rotary F-ATP Synthases |
| title_sort | structural asymmetry and kinetic limping of single rotary f atp synthases |
| topic | F<sub>O</sub>F<sub>1</sub> ATP synthase <i>Escherichia coli</i> single-molecule fluorescence symmetry cryo-EM structure subunit rotation elasticity |
| url | https://www.mdpi.com/1420-3049/24/3/504 |
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