Single-molecule FRET combined with electrokinetic trapping reveals real-time enzyme kinetics of individual F-ATP synthases

Sielaff, Hendrik; Dienerowitz, Frank; Dienerowitz, Maria

doi:10.1039/d1nr05754e

Cited by 4 publications

(3 citation statements)

References 61 publications

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“…The reader is referred to previous publications for a full description of the ABEL trap system. 16,25,26 The sample solution containing 150 pM of labelled Rep (Alexa Fluor 546 as the donor, Alexa Fluor 647 as the acceptor) was loaded into a microfluidic chip with the molecule concentration adjusted to monitor five or less molecules per second. In the absence of trapping, the molecules diffused freely through the single beam excitation volume and were detected with a confocal setup.…”

Section: Resultsmentioning

confidence: 99%

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Single-molecule FRET dynamics of molecular motors in an anti-Brownian electrokinetic trap

Sielaff

Howard²,

Quinn³

et al. 2023

Frontiers in Biophotonics and Imaging II

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Monitoring the dynamic behaviour of individual molecular motors in solution reveals insights into their catalytic activities. Single-molecule Förster resonance energy transfer (smFRET) allows us to study molecular motors in real time at high spatial and temporal resolution. Combining smFRET with molecular confinement enables us to increase the observation time up to several seconds without tethered surface attachment of the observed molecules. Here, we employed an Anti-Brownian ELectrokinetic trap (ABEL trap) that used variable homogeneous electric fields to actively confine single molecules to a femtolitre sized observation volume by acting on their surface charge. We present a non-invasive confinement method that allows trapping of molecular motors like the Rep helicase and the F O F 1 -ATP synthase. In Escherichia coli the ATP-dependent Rep DNA helicase facilitates the replisome progression and is necessary for the restart of the DNA replication machinery. We selectively trapped active Rep molecules based on their smFRET signal with sub-millisecond temporal resolution recording conformational switching events during observation times of up to several seconds. In addition, we observed the ATP-hydrolysis driven rotation of individual F O F 1 -ATP synthase molecules over numerous consecutive reaction cycles and extracted their kinetic rates.

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Section: Resultsmentioning

confidence: 99%

“…Trapping and monitoring such a liposome-bound FRETlabelled F O F 1 -ATP synthase with the ABEL trap enabled us to observe rotational rates of E. coli F O F 1 -ATP synthase molecules during hydrolysis depending on ATP concentrations. 25 We were able to track individual molecular motors in real time with millisecond time resolution.…”

Section: Introductionmentioning

confidence: 99%

Single-molecule FRET dynamics of molecular motors in an anti-Brownian electrokinetic trap

Sielaff

Howard²,

Quinn³

et al. 2023

Frontiers in Biophotonics and Imaging II

Self Cite

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“…X-ray can solve structures up to 4000 kD (PDB website statistics). FRET is used for medium-sized proteins but has been reported for up to a 540 kD protein (Sielaff et al ., 2022). …”

Section: Part 2: Computational Modeling and Characterization Of Mapidsmentioning

confidence: 99%

Myofilament-associated proteins with intrinsic disorder (MAPIDs) and their resolution by computational modeling

Sun

Kekenes‐Huskey

2023

Quart. Rev. Biophys.

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The cardiac sarcomere is a cellular structure in the heart that enables muscle cells to contract. Dozens of proteins belong to the cardiac sarcomere, which work in tandem to generate force and adapt to demands on cardiac output. Intriguingly, the majority of these proteins have significant intrinsic disorder that contributes to their functions, yet the biophysics of these intrinsically disordered regions (IDRs) have been characterized in limited detail. In this review, we first enumerate these myofilament-associated proteins with intrinsic disorder (MAPIDs) and recent biophysical studies to characterize their IDRs. We secondly summarize the biophysics governing IDR properties and the state-of-the-art in computational tools toward MAPID identification and characterization of their conformation ensembles. We conclude with an overview of future computational approaches toward broadening the understanding of intrinsic disorder in the cardiac sarcomere.

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An FOF1-ATPase motor-embedded chromatophore as a nanorobot for overcoming biological barriers and targeting acidic tumor sites

Yang,

Zhou,

Lou

et al. 2024

Acta Biomaterialia

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Single-molecule FRET combined with electrokinetic trapping reveals real-time enzyme kinetics of individual F-ATP synthases

Abstract: Combining smFRET and electrokinetic trapping to investigate dynamic conformational changes of single molecules in solution with millisecond time resolution for observation times extending beyond the duration of several reaction cycles.

Cited by 4 publications

References 61 publications

Single-molecule FRET dynamics of molecular motors in an anti-Brownian electrokinetic trap

Single-molecule FRET dynamics of molecular motors in an anti-Brownian electrokinetic trap

Myofilament-associated proteins with intrinsic disorder (MAPIDs) and their resolution by computational modeling

An FOF1-ATPase motor-embedded chromatophore as a nanorobot for overcoming biological barriers and targeting acidic tumor sites

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