Conformational dynamics of the rotary subunit F in the A3B3<scp>DF</scp> complex of Methanosarcina mazei Gö1 A‐<scp>ATP</scp> synthase monitored by single‐molecule <scp>FRET</scp>

Singh, Dhirendra; Sielaff, Hendrik; Börsch, Michael; Grüber, Gerhard

doi:10.1002/1873-3468.12605

Cited by 10 publications

(13 citation statements)

References 38 publications

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“…He noticed that the release of ATP, but not its formation, requires energy input. Since then, the mechanism has been confirmed by a series of single molecule rotation experiments, first with the F-ATP synthase [56,63,64,65,66,67,68,69,70,71], and subsequently with the A-ATP synthase and the V-ATPase [72,73,74,75,76,77]. During ATP hydrolysis, the counterclockwise (CCW) rotation (when viewed from the membrane side) of the central stalk (γε c 10 ) progresses in three major steps of 120°.…”

Section: Introductionmentioning

confidence: 99%

Structural Asymmetry and Kinetic Limping of Single Rotary F-ATP Synthases

et al. 2019

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F-ATP synthases use proton flow through the FO domain to synthesize ATP in the F1 domain. In Escherichia coli, the enzyme consists of rotor subunits γεc10 and stator subunits (αβ)3δab2. Subunits c10 or (αβ)3 alone are rotationally symmetric. However, symmetry is broken by the b2 homodimer, which together with subunit δa, forms a single eccentric stalk connecting the membrane embedded FO domain with the soluble F1 domain, and the central rotating and curved stalk composed of subunit γε. Although each of the three catalytic binding sites in (αβ)3 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 b2δ in F1 and with b2a in FO. 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.

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Section: Introductionmentioning

confidence: 99%

Structural Asymmetry and Kinetic Limping of Single Rotary F-ATP Synthases

et al. 2019

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“…Protein mutagenesis, production, and purification started out from a purchased pQE-T7 expression vector harboring the gene encoding for ACT (UNIPROT P01011; Qiagen). CD measurements were recorded on a Jasco J-815 spectropolarimeter, ITC experiments were performed in a Nano ITC standard cell (TA Instruments) and fluorescence measurements were obtained with a custom-designed confocal microscope modified to record doxorubicin fluorescence for λ >605 nm (28). Diffraction data were recorded at synchrotron beamlines BL14.1 and BL14.2 of BESSY II (Berlin, Germany) and beamline P14 of PETRA III (Hamburg, Germany).…”

Section: Methodsmentioning

confidence: 99%

Design of an allosterically modulated doxycycline and doxorubicin drug-binding protein

Schmidt¹,

Gardill²,

Kern³

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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The allosteric interplay between distant functional sites present in a single protein provides for one of the most important regulatory mechanisms in biological systems. While the design of ligand-binding sites into proteins remains challenging, this holds even truer for the coupling of a newly engineered binding site to an allosteric mechanism that regulates the ligand affinity. Here it is shown how computational design algorithms enabled the introduction of doxycycline- and doxorubicin-binding sites into the serine proteinase inhibitor (serpin) family member α1-antichymotrypsin. Further engineering allowed exploitation of the proteinase-triggered serpin-typical S-to-R transition to modulate the ligand affinities. These design variants follow strategies observed in naturally occurring plasma globulins that allow for the targeted delivery of hormones in the blood. By analogy, we propose that the variants described in the present study could be further developed to allow for the delivery of the antibiotic doxycycline and the anticancer compound doxorubicin to tissues/locations that express specific proteinases, such as bacterial infection sites or tumor cells secreting matrix metalloproteinases.

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“…For time-resolved single-molecule anisotropy and smFRET measurements in solution (i.e. homoFRET), a custom-designed confocal microscope was used based on an Olympus IX71 [10, 20-25]. Ps-pulsed linearly polarized excitation was provided with 488 nm at 80 MHz (PicoTa 490, Picoquant) using a 60x water immersion objective (N.A.…”

Section: Methodsmentioning

confidence: 99%

“…Using single-molecule fluorescence methods we aim at unravelling the conformational dynamics of NTSR1 with respect to ligand binding, and G protein interaction, and how a membrane potential possibly influences the receptor activity. We have previously built confocal microscopes [10, 11] to observe individual membrane proteins one-after-another by single-molecule Förster resonance energy transfer (smFRET). Our current study extends smFRET measurements to the NTSR1 system.…”

Section: Introductionmentioning

confidence: 99%

Towards monitoring conformational changes of the GPCR neurotensin receptor 1 by single-molecule FRET

Heitkamp

Grisshammer

Börsch

2018

Multiphoton Microscopy in the Biomedical Sciences XVIII

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Neurotensin receptor 1 (NTSR1) is a G protein-coupled receptor that is important for signaling in the brain and the gut. Its agonist ligand neurotensin (NTS), a 13-amino-acid peptide, binds with nanomolar affinity from the extracellular side to NTSR1 and induces conformational changes that trigger intracellular signaling processes. Our goal is to monitor the conformational dynamics of single fluorescently labeled NTSR1. For this, we fused the fluorescent protein mNeonGreen to the C terminus of NTSR1, purified the receptor fusion protein from membranes, and reconstituted NTSR1 into liposomes with polar lipids. Using single-molecule anisotropy measurements, NTSR1 was found to be monomeric in liposomes, with a small fraction being dimeric and oligomeric, showing homoFRET. Similar results were obtained for NTSR1 in detergent solution. Furthermore, we demonstrated agonist binding to NTSR1 by time-resolved single-molecule Förster resonance energy transfer (smFRET), using neurotensin labeled with the fluorophore ATTO594.

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Conformational dynamics of the rotary subunit F in the A₃B₃DF complex of Methanosarcina mazei Gö1 A‐ATP synthase monitored by single‐molecule FRET

Cited by 10 publications

References 38 publications

Structural Asymmetry and Kinetic Limping of Single Rotary F-ATP Synthases

Structural Asymmetry and Kinetic Limping of Single Rotary F-ATP Synthases

Design of an allosterically modulated doxycycline and doxorubicin drug-binding protein

Towards monitoring conformational changes of the GPCR neurotensin receptor 1 by single-molecule FRET

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Conformational dynamics of the rotary subunit F in the A3B3DF complex of Methanosarcina mazei Gö1 A‐ATP synthase monitored by single‐molecule FRET

Cited by 10 publications

References 38 publications

Structural Asymmetry and Kinetic Limping of Single Rotary F-ATP Synthases

Structural Asymmetry and Kinetic Limping of Single Rotary F-ATP Synthases

Design of an allosterically modulated doxycycline and doxorubicin drug-binding protein

Towards monitoring conformational changes of the GPCR neurotensin receptor 1 by single-molecule FRET

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Product

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Conformational dynamics of the rotary subunit F in the A₃B₃DF complex of Methanosarcina mazei Gö1 A‐ATP synthase monitored by single‐molecule FRET