Abstract
The circadian clock proteins KaiA, KaiB, and KaiC reconstitute a remarkable circa-24 h oscillation of KaiC phosphorylation that persists for many days in vitro. Here we use high-speed atomic force microscopy (HS-AFM) to visualize in real time and quantify the dynamic interactions of KaiA with KaiC on sub-second timescales. KaiA transiently interacts with KaiC, thereby stimulating KaiC autokinase activity. As KaiC becomes progressively more phosphorylated, KaiA’s affinity for KaiC weakens, revealing a feedback of KaiC phosphostatus back onto the KaiA-binding events. These non-equilibrium interactions integrate high-frequency binding and unbinding events, thereby refining the period of the longer term oscillations. Moreover, this differential affinity phenomenon broadens the range of Kai protein stoichiometries that allow rhythmicity, explaining how the oscillation is resilient in an in vivo milieu that includes noise. Therefore, robustness of rhythmicity on a 24-h scale is explainable by molecular events occurring on a scale of sub-seconds.
Highlights
The circadian clock proteins KaiA, KaiB, and KaiC reconstitute a remarkable circa-24 h oscillation of KaiC phosphorylation that persists for many days in vitro
phosphoform dependent differential affinity (PDDA) has broad consequences that were not anticipated by previous modeling and demonstrates an exquisite interplay of mechanisms that enable oscillatory resilience to changes in clock protein concentrations that are likely to occur in vivo[3,20]
We computationally and experimentally test PDDAconferred resilience by in silico simulations and by biochemical manipulation of the in vitro KaiABC oscillation. These results indicate how a model biochemical oscillator is able to integrate high-frequency molecular events into a circadian timekeeper in environments of fluctuating clock protein levels resulting from molecular noise
Summary
The circadian clock proteins KaiA, KaiB, and KaiC reconstitute a remarkable circa-24 h oscillation of KaiC phosphorylation that persists for many days in vitro. The process of testing a model for the intermolecular synchronization of KaiC phosphostatus in the in vitro KaiABC reaction[22] led us to serendipitously discover dynamic highfrequency interactions between KaiA and KaiC that explain how a broader range of KaiA:KaiC stoichiometry is permissive This result is unexpected because KaiA interacts with KaiC hexamers during the phosphorylation half-cycle phase of the in vitro oscillator repeatedly on and off on the timescale of fractions of seconds; this is a timescale that has been ignored in previous analyses of the KaiABC oscillator because interactions of this frequency are not intuitively expected to help explain a biochemical oscillation of circa-24 hours. These results indicate how a model biochemical oscillator is able to integrate high-frequency molecular events into a circadian timekeeper in environments of fluctuating clock protein levels resulting from molecular noise
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