The circadian system of the cyanobacterium
PCC 7942 relies on a three-protein nanomachine (KaiA, KaiB, and KaiC) that undergoes an oscillatory phosphorylation cycle with a period of ~24 h. This core ...oscillator can be reconstituted in vitro and is used to study the molecular mechanisms of circadian timekeeping and entrainment. Previous studies showed that two key metabolic changes that occur in cells during the transition into darkness, changes in the ATP/ADP ratio and redox status of the quinone pool, are cues that entrain the circadian clock. By changing the ATP/ADP ratio or adding oxidized quinone, one can shift the phase of the phosphorylation cycle of the core oscillator in vitro. However, the in vitro oscillator cannot explain gene expression patterns because the simple mixture lacks the output components that connect the clock to genes. Recently, a high-throughput in vitro system termed the in vitro clock (IVC) that contains both the core oscillator and the output components was developed. Here, we used IVC reactions and performed massively parallel experiments to study entrainment, the synchronization of the clock with the environment, in the presence of output components. Our results indicate that the IVC better explains the in vivo clock-resetting phenotypes of wild-type and mutant strains and that the output components are deeply engaged with the core oscillator, affecting the way input signals entrain the core pacemaker. These findings blur the line between input and output pathways and support our previous demonstration that key output components are fundamental parts of the clock.
Circadian clocks control gene expression to provide an internal representation of local time. We report reconstitution of a complete cyanobacterial circadian clock in vitro, including the central ...oscillator, signal transduction pathways, downstream transcription factor, and promoter DNA. The entire system oscillates autonomously and remains phase coherent for many days with a fluorescence-based readout that enables real-time observation of each component simultaneously without user intervention. We identified the molecular basis for loss of cycling in an arrhythmic mutant and explored fundamental mechanisms of timekeeping in the cyanobacterial clock. We find that SasA, a circadian sensor histidine kinase associated with clock output, engages directly with KaiB on the KaiC hexamer to regulate period and amplitude of the central oscillator. SasA uses structural mimicry to cooperatively recruit the rare, fold-switched conformation of KaiB to the KaiC hexamer to form the nighttime repressive complex and enhance rhythmicity of the oscillator, particularly under limiting concentrations of KaiB. Thus, the expanded in vitro clock reveals previously unknown mechanisms by which the circadian system of cyanobacteria maintains the pace and rhythmicity under variable protein concentrations.
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 C6 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.
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.
Circadian clocks are ubiquitous timing systems that induce rhythms of biological activities in synchrony with night and day. In cyanobacteria, timing is generated by a posttranslational clock ...consisting of KaiA, KaiB, and KaiC proteins and a set of output signaling proteins, SasA and CikA, which transduce this rhythm to control gene expression. Here, we describe crystal and nuclear magnetic resonance structures of KaiB-KaiC, KaiA-KaiB-KaiC, and CikA-KaiB complexes. They reveal how the metamorphic properties of KaiB, a protein that adopts two distinct folds, and the post–adenosine triphosphate hydrolysis state of KaiC create a hub around which nighttime signaling events revolve, including inactivation of KaiA and reciprocal regulation of the mutually antagonistic signaling proteins, SasA and CikA.
In the present research, Proton conducting Ceramic electrolyte Fuel Cell (PCFC) has been fabricated with BaZr0.4Ce0.45Y0.15O3−δ electrolyte, NiO–BaZr0.4Ce0.45Y0.15O3−δ as anode and ...Ba0.5Sr0.5Co0.8Fe0.2O3−δ as a cathode material. The effects of anodic and cathodic gas flow rates and partial pressures on PCFC performance are studied by varying the gas flow rates between 50 and 200sccm while partial pressures between 0.25 and 1atm at one electrode by keeping constant gas flow rate and partial pressure at another electrode. The different electrode processes occurring at the electrodes and electrode/electrolyte interfaces are analyzed by electrochemical impedance spectroscopy. It is observed that gas flow rate doesn’t affect more on the fuel cell performance while increase in gas partial pressure changes the performance of PCFC. The variation in cathodic pO2 shows that the oxygen dissociation and charge transfer at cathode electrode are the major contributors toward overall electrode polarization resistance. A peak power density of ~0.80W/cm−2 is achieved at 700°C with 200sccm of wet H2 and air. The stability of the PCFC is also studied under CO2 condition and it has shown stable performance without any degradation.
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.
Synopsis
This study takes a data‐driven kinetic modeling approach to characterize the interaction between KaiA and KaiC in the cyanobacterial circadian clock to understand how the oscillator responds to changes in cellular metabolic conditions.
An extensive dataset of KaiC autophosphorylation measurements is generated and used to constrain a detailed yet mechanistically naive kinetic model within a Bayesian parameter estimation framework.
KaiA concentration tunes the sensitivity of KaiC autophosphorylation and the period of the full oscillator to %ATP.
The model reveals an ultrasensitive dependence of KaiC phosphorylation on KaiA concentration as a result of differential KaiA binding affinity to ADP‐ vs. ATP‐bound KaiC.
Ultrasensitivity in KaiC phosphorylation likely contributes to metabolic compensation by suppressing premature phosphorylation at nighttime.
This study takes a data‐driven kinetic modeling approach to characterize the interaction between KaiA and KaiC in the cyanobacterial circadian clock to understand how the oscillator responds to changes in cellular metabolic conditions.
This work demonstrates that all packing in α-helices can be simplified to repetitive patterns of a single motif: the knob–socket. Using the precision of Voronoi Polyhedra/Delauney Tessellations to ...identify contacts, the knob–socket is a four-residue tetrahedral motif: a knob residue on one α-helix packs into the three-residue socket on another α-helix. The principle of the knob–socket model relates the packing between levels of protein structure: the intra-helical packing arrangements within secondary structure that permit inter-helix tertiary packing interactions. Within an α-helix, the three-residue sockets arrange residues into a uniform packing lattice. Inter-helix packing results from a definable pattern of interdigitated knob–socket motifs between two α-helices. Furthermore, the knob–socket model classifies three types of sockets: (1) free, favoring only intra-helical packing; (2) filled, favoring inter-helical interactions; and (3) non, disfavoring α-helical structure. The amino acid propensities in these three socket classes essentially represent an amino acid code for structure in α-helical packing. Using this code, we used a novel yet straightforward approach for the design of α-helical structure to validate the knob–socket model. Unique sequences for three peptides were created to produce a predicted amount of α-helical structure: mostly helical, some helical, and no helix. These three peptides were synthesized, and helical content was assessed using CD spectroscopy. The measured α-helicity of each peptide was consistent with the expected predictions. These results and analysis demonstrate that the knob–socket motif functions as the basic unit of packing and presents an intuitive tool to decipher the rules governing packing in protein structure.
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► Knob-socket model provides a specific code for α-helical packing structure. ► Knob-socket motif forms the basic unit of protein packing. ► New paradigm defines the 2° structure packing that allows 3° and 4° interactions. ► Simple patterns of knob–socket describe all helix packing. ► New robust approach to design of alpha-helix structures.
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