Real-Time In Vitro Fluorescence Anisotropy of the Cyanobacterial Circadian Clock

Heisler, Joel; Chavan, Archana G.; Chang, Yong Gang; LiWang, Andy

doi:10.3390/mps2020042

Cited by 19 publications

(24 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Such a scenario might allow the [KaiA]/[KaiC] ratio to be an important parameter in vivo for adjusting the sensitivity of the clock to the daily metabolic rhythm of the cell. To address this question, we characterized the dependence of the period of the in vitro KaiABC oscillator on [KaiA] and %ATP using a fluorescence polarization assay (Leypunskiy et al , ; Heisler et al , ) (Figs D and EV3A and B). Consistent with the hypothesis, we found that low KaiA concentration enhances the period sensitivity to %ATP compared to the standard condition (1.5 μM KaiA).…”

Section: Resultsmentioning

confidence: 99%

Bayesian modeling reveals metabolite‐dependent ultrasensitivity in the cyanobacterial circadian clock

Lavrentovich

Chavan

et al. 2020

Molecular Systems Biology

Self Cite

View full text Add to dashboard Cite

Mathematical models can enable a predictive understanding of mechanism in cell biology by quantitatively describing complex networks of interactions, but such models are often poorly constrained by available data. Owing to its relative biochemical simplicity, the core circadian oscillator in Synechococcus elongatus has become a prototypical system for studying how collective dynamics emerge from molecular interactions. The oscillator consists of only three proteins, KaiA, KaiB, and KaiC, and near-24-h cycles of KaiC phosphorylation can be reconstituted in vitro. Here, we formulate a molecularly detailed but mechanistically naive model of the KaiA-KaiC subsystem and fit it directly to experimental data within a Bayesian parameter estimation framework. Analysis of the fits consistently reveals an ultrasensitive response for KaiC phosphorylation as a function of KaiA concentration, which we confirm experimentally. This ultrasensitivity primarily results from the differential affinity of KaiA for competing nucleotide-bound states of KaiC. We argue that the ultrasensitive stimulus-response relation likely plays an important role in metabolic compensation by suppressing premature phosphorylation at nighttime.

show abstract

Section: Resultsmentioning

confidence: 99%

Bayesian modeling reveals metabolite‐dependent ultrasensitivity in the cyanobacterial circadian clock

Lavrentovich

Chavan

et al. 2020

Molecular Systems Biology

Self Cite

View full text Add to dashboard Cite

show abstract

“…To more accurately measure the scaling of entrained phase with day length in KaiABC oscillator reactions, we turned to a fluorescence polarization probe that enables automated measurement of oscillator state with high temporal resolution ( Figure 2—figure supplement 1 ) ( Chang et al, 2012 ; Heisler et al, 2017 ). We used this assay to determine clock phases in free-running conditions after entraining the Kai proteins with three metabolic cycles, analogous to the design of the in vivo experiments.…”

Section: Resultsmentioning

confidence: 99%

The cyanobacterial circadian clock follows midday in vivo and in vitro

Leypunskiy

Lin

Yoo

et al. 2017

eLife

View full text Add to dashboard Cite

Circadian rhythms are biological oscillations that schedule daily changes in physiology. Outside the laboratory, circadian clocks do not generally free-run but are driven by daily cues whose timing varies with the seasons. The principles that determine how circadian clocks align to these external cycles are not well understood. Here, we report experimental platforms for driving the cyanobacterial circadian clock both in vivo and in vitro. We find that the phase of the circadian rhythm follows a simple scaling law in light-dark cycles, tracking midday across conditions with variable day length. The core biochemical oscillator comprised of the Kai proteins behaves similarly when driven by metabolic pulses in vitro, indicating that such dynamics are intrinsic to these proteins. We develop a general mathematical framework based on instantaneous transformation of the clock cycle by external cues, which successfully predicts clock behavior under many cycling environments.DOI: http://dx.doi.org/10.7554/eLife.23539.001

show abstract

“…However, the discontinuous nature of these assays meant that multiple samples had to be prepared to provide time-domain information. More recently, fluorescence correlation spectroscopy (FCS) and fluorescence polarization/anisotropy (FA) have been employed to study KaiB binding by fluorophore tagging, producing tagged proteins using either cell free synthesis , or site-directed mutagenesis. , Fluorescence assays are an improvement to previous discontinuous methods as the fluorescence signals could be continuously monitored provided the fluorophore tags are not photobleached. However, the myriad of stoichiometries existing in Kai reaction mixtures, tetrameric and monomeric KaiB and (sub)stoichiometric KaiBC and KaiABC complexes, ,, invalidate models that assume two species with differing molecular weights.…”

mentioning

confidence: 99%

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

et al. 2020

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Real-Time In Vitro Fluorescence Anisotropy of the Cyanobacterial Circadian Clock

Cited by 19 publications

References 21 publications

Bayesian modeling reveals metabolite‐dependent ultrasensitivity in the cyanobacterial circadian clock

Bayesian modeling reveals metabolite‐dependent ultrasensitivity in the cyanobacterial circadian clock

The cyanobacterial circadian clock follows midday in vivo and in vitro

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

Contact Info

Product

Resources

About