Quantitative EPR — Sensitivity to experimental conditions and optimal setting of recording parameters

Czoch, R.

doi:10.1007/bf03163115

Cited by 17 publications

(12 citation statements)

References 25 publications

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“…Double integration-based normalization was avoided due to known numerical instability and susceptibility to noise (see Figure S10a). Instead, the spectra were normalized by using the real-time kinetic data of KaiB in the presence of a 10-fold excess of KaiC EE . The real-time data can be written as where θ is the scaling factor to scale B free .…”

Section: Materials and Methodsmentioning

confidence: 99%

Monitoring Protein–Protein Interactions in the Cyanobacterial Circadian Clock in Real Time via Electron Paramagnetic Resonance Spectroscopy

et al. 2020

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The cyanobacterial circadian clock in Synechococcus elongatus consists of three proteins, KaiA, KaiB, and KaiC. KaiA and KaiB rhythmically interact with KaiC to generate stable oscillations of KaiC phosphorylation with a period of 24 h. The observation of stable circadian oscillations when the three clock proteins are reconstituted and combined in vitro makes it an ideal system for understanding its underlying molecular mechanisms and circadian clocks in general. These oscillations were historically monitored in vitro by gel electrophoresis of reaction mixtures based on the differing electrophoretic mobilities between various phosphostates of KaiC. As the KaiC phospho-distribution represents only one facet of the oscillations, orthogonal tools are necessary to explore other interactions to generate a full description of the system. However, previous biochemical assays are discontinuous or qualitative. To circumvent these limitations, we developed a spin-labeled KaiB mutant that can differentiate KaiC-bound KaiB from free KaiB using continuous-wave electron paramagnetic resonance spectroscopy that is minimally sensitive to KaiA. Similar to wild-type (WT-KaiB), this labeled mutant, in combination with KaiA, sustains robust circadian rhythms of KaiC phosphorylation. This labeled mutant is hence a functional surrogate of WT-KaiB and thus participates in and reports on autonomous macroscopic circadian rhythms generated by mixtures that include KaiA, KaiC, and ATP. Quantitative kinetics could be extracted with improved precision and time resolution. We describe design principles, data analysis, and limitations of this quantitative binding assay and discuss future research necessary to overcome these challenges.

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Section: Materials and Methodsmentioning

confidence: 99%

Monitoring Protein–Protein Interactions in the Cyanobacterial Circadian Clock in Real Time via Electron Paramagnetic Resonance Spectroscopy

et al. 2020

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“…Quantitative ESR. 19 All measurements were carried out on a Bruker ER200D SRC spectrometer and/or a Bruker ESP 300E, operating with an X-band standard cavity (ν ca. 9.4 GHz) and interfaced to a Bruker Aspect 3000 data system.…”

Section: Methodsmentioning

confidence: 99%

Nitroxide-Mediated Living Radical Polymerization: Determination of the Rate Coefficient for Alkoxyamine C−O Bond Homolysis by Quantitative ESR

1999

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The rate coefficient for alkoxyamine C−O bond homolysis has been determined over a range of temperatures for both 2-tert-butoxy-1-phenyl-1-(1-oxy-2,2,6,6-tetramethylpiperidinyl)ethane (1) and a polystyrene−TEMPO (approximately 75 units) adduct using quantitative ESR. In a thermostated solution of the alkoxyamine species in toluene the nitroxide concentration was monitored via ESR. Measurements were performed under atmospheric conditions to create pseudo-first-order kinetics with oxygen to serve as a scavenger for the carbon-centered radicals. The acquired kinetic data for alkoxyamine 1 were compared with data obtained via nitroxide-exchange experiments using HPLC to monitor the alkoxyamine concentrations. The data for the polystyrene alkoxyamine were compared with literature data obtained by Fukuda via his GPC method. The quantitative ESR measurements proved to be an easy and more important an accurate method to determine values for the rate coefficient for alkoxyamine C−O bond homolysis. The Arrhenius parameters obtained via this method were A = 1.10 × 1014 s-1 and E act = 133.2 kJ mol-1 for alkoxyamine 1 and A = 1.02 × 1016 s-1 and E act = 140.9 kJ mol-1 for its polymeric analogue.

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“…As KaiC is a member of the AAA+ ATPase family, 42 many of which are hexameric but capable of breaking their C 6 symmetry by means of differential nucleotide incorporation, 43 inter-KaiB cooperativity is likely tuned by KaiC phosphostate distribution within individual KaiC hexamers. Asymmetry in KaiC can then be introduced by intrinsic ATPase activity of CI 12 and/or nucleotide exchange in CII, 8 both of which are modulated by KaiC phosphostate. 38 Nucleotide exchange in CII has recently been modeled by molecular dynamics simulations to result in a split washer structure, with this asymmetry propagating toward CI.…”

Section: ■ Discussionmentioning

confidence: 99%

“…At night, S431 phosphorylation in KaiC leads to changes in the flexibility of the C-terminal domain of KaiC (CII) and subsequent CII-CI ring−ring stacking (CI, Nterminal domain of KaiC). 10,11 This stacking interaction, along with ADP binding in CI enabled by CI ATPase activity, 12,13 biases KaiC toward an alternative conformation that is KaiBbinding competent 10,14 at the loop region of the N-terminal domain (B-loops). 15 This KaiB-binding competent KaiC state stabilizes a fold-switched conformer of KaiB (fs-KaiB) 16 that is capable of sequestering KaiA into a ternary KaiABC complex.…”

Section: ■ Introductionmentioning

confidence: 99%

A Night-Time Edge Site Intermediate in the Cyanobacterial Circadian Clock Identified by EPR Spectroscopy

et al. 2022

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As the only circadian oscillator that can be reconstituted in vitro with its constituent proteins KaiA, KaiB, and KaiC using ATP as an energy source, the cyanobacterial circadian oscillator serves as a model system for detailed mechanistic studies of day−night transitions of circadian clocks in general. The day-to-night transition occurs when KaiB forms a night-time complex with KaiC to sequester KaiA, the latter of which interacts with KaiC during the day to promote KaiC autophosphorylation. However, how KaiB forms the complex with KaiC remains poorly understood, despite the available structures of KaiB bound to hexameric KaiC. It has been postulated that KaiB-KaiC binding is regulated by inter-KaiB cooperativity. Here, using spin labeling continuous-wave electron paramagnetic resonance spectroscopy, we identified and quantified two subpopulations of KaiC-bound KaiB, corresponding to the "bulk" and "edge" KaiBC sites in stoichiometric and substoichiometric KaiB i C 6 complexes (i = 1−5). We provide kinetic evidence to support the intermediacy of the "edge" KaiBC sites as bridges and nucleation sites between free KaiB and the "bulk" KaiBC sites. Furthermore, we show that the relative abundance of "edge" and "bulk" sites is dependent on both KaiC phosphostate and KaiA, supporting the notion of phosphorylation-state controlled inter-KaiB cooperativity. Finally, we demonstrate that the interconversion between the two subpopulations of KaiC-bound KaiB is intimately linked to the KaiC phosphorylation cycle. These findings enrich our mechanistic understanding of the cyanobacterial clock and demonstrate the utility of EPR in elucidating circadian clock mechanisms.

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Quantitative EPR — Sensitivity to experimental conditions and optimal setting of recording parameters

Cited by 17 publications

References 25 publications

Monitoring Protein–Protein Interactions in the Cyanobacterial Circadian Clock in Real Time via Electron Paramagnetic Resonance Spectroscopy

Monitoring Protein–Protein Interactions in the Cyanobacterial Circadian Clock in Real Time via Electron Paramagnetic Resonance Spectroscopy

Nitroxide-Mediated Living Radical Polymerization: Determination of the Rate Coefficient for Alkoxyamine C−O Bond Homolysis by Quantitative ESR

A Night-Time Edge Site Intermediate in the Cyanobacterial Circadian Clock Identified by EPR Spectroscopy

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