Conversion between two conformational states of KaiC is induced by ATP hydrolysis as a trigger for cyanobacterial circadian oscillation

Abstract

The cyanobacterial circadian oscillator can be reconstituted in vitro by mixing three clock proteins, KaiA, KaiB and KaiC, with ATP. KaiC is the only protein with circadian rhythmic activities. In the present study, we tracked the complex formation of the three Kai proteins over time using blue native (BN) polyacrylamide gel electrophoresis (PAGE), in which proteins are charged with the anionic dye Coomassie brilliant blue (CBB). KaiC was separated as three bands: the KaiABC complex, KaiC hexamer and KaiC monomer. However, no KaiC monomer was observed using gel filtration chromatography and CBB-free native PAGE. These data indicate two conformational states of KaiC hexamer and show that the ground-state KaiC (gs-KaiC) is stable and competent-state KaiC (cs-KaiC) is labile and degraded into monomers by the binding of CBB. Repeated conversions from gs-KaiC to cs-KaiC were observed over 24 h using an in vitro reconstitution system. Phosphorylation of KaiC promoted the conversion from gs-KaiC to cs-KaiC. KaiA sustained the gs-KaiC state, and KaiB bound only cs-KaiC. An E77Q/E78Q-KaiC variant that lacked N-terminal ATPase activity remained in the gs-KaiC state. Taken together, ATP hydrolysis induces the formation of cs-KaiC and promotes the binding of KaiB, which is a trigger for circadian oscillations.

Atomic force microscopy analysis of SasA‐KaiC complex formation involved in information transfer from the KaiABC clock machinery to the output pathway in cyanobacteria

“…These results suggest that KaiC WT 6mer was more stable than KaiC DD 6mer . It is consistent with a previous report showing that nonphosphorylated KaiC 6mer was more stable than phosphorylated KaiC 6mer (Oyama, Azai, Nakamura, Tanaka, & Terauchi, 2016).…”

Analysis of the degradation of amyloid beta fibrils after separation via the combination of non‐denaturing agarose electrophoresis and Congo red dye staining

“…The digestion of 0.23 nmol of the Aβ fibrils using 22.5 pmol of nattokinase at 37°C, exhibited a time‐dependent increase up to 6 h, beyond which the effect of time remained insignificant (Figure b). In addition to nattokinase, neprilysin and matrix metalloprotease also degrade the Aβ fibrils . Thus, these enzymes are applicable for the degradation of Aβ fibrils in this method.…”

“…Figure 3 displays changes in the Aβ 42 fibrils content at the cathode end of the non-denaturing agarose gel electrophoresis for: (a) 0.23 nmol of Aβ 42 fibrils incubated with 0, 4.5, 9.0, 22.5, and 45 pmol of nattokinase at 37 • C for 24 h, and (Figure 3b). In addition to nattokinase, neprilysin and matrix metalloprotease also degrade the Aβ fibrils [12][13][14]. Thus, these enzymes are applicable for the degradation of Aβ fibrils in this method.…”

Phosphorylation at Thr432 induces structural destabilization of the CII ring in the circadian oscillator KaiC

“…Plasmids for the overexpression of the two CII KaiC variants, CII KaiC DE and CII KaiC AA , were constructed independently of the plasmid for CII KaiC as follows. The region corresponding to the KaiC CII domain (Leu249‐Ser519) was amplified by PCR using two plasmids as templates that were used for the overexpression of KaiC‐DE and KaiC‐AA proteins in the previous work . The PCR fragments were cloned into the Bsa I site of pASK‐IBA‐5plus to overexpress CII KaiC DE and CII KaiC AA as N‐terminal Strep‐tag fusion proteins.…”