The influence of KaiA mutations on its function in the KaiABC circadian clock system

The core oscillator of the circadian clock of cyanobacteria consists of three proteins, KaiA, KaiB, and KaiC. The KaiABC oscillator can be re-constituted in vitro with the purified proteins in buffer containing ATP and Mg2+. The interaction between KaiA and KaiC has not been well studied. In this article, different KaiA mutants were designed and used to elucidate the influence of KaiA structure on its function in the in vitro system. Molecular dynamics simulations were adopted to study the structural flexibility of KaiA homodimer. The data presented in this article provide further experimental supports to our work in Chen et al. (2017) [1].


How data was acquired
The elution profiles of the proteins were collected using a ÄKTA Purifier 100 (GE) system. The images of the SDS-PAGE gels were taken on a gel imaging system (Kodak, Gel Logic 200).The molecular dynamics simulations were performed in NAMD on a GPU server.

Data format
Raw, analyzed Experimental factors The clock proteins were expressed in E. coli and purified. Then the proteins were mixed under different combinations and incubated at 30°C to collect samples at indicated time points. The samples were analyzed with 8% SDS-PAGE gels to analyze the phosphorylation of KaiC.

Experimental features
Test KaiA's function using SDS-PAGE and molecular dynamics simulations Data source location College of Medical Science, China Three Gorges University, Yichang, China Data accessibility Data are presented in the article

Value of the data
This data article presents versatile protein design strategies to study the structure-function relationship of KaiA.
The data article focused on the analysis of the in vitro KaiABC system using SDS-PAGE. This dataset could be a useful reference to study the relationship between protein structural flexibility and functional dynamics.

Data
Circadian rhythms are the~24 h cycles of the physiological processes in living beings on Earth. The cyanobacterial circadian clock consists three proteins, namely KaiA, KaiB, and KaiC. KaiA binds to KaiC through the interaction between KaiA's C-terminal domains and KaiC's C-terminal tail [2]. However, the details of KaiA exerts its function are still not known very well, especially how KaiA does functional switch between active and in-active. Very recently, Tseng et al. presented a possible mechanism that KaiA gets auto-inhibited [3]. Our work in this article and in Ref. [1] provided further evidence and clues to the function regulation of KaiA.
Using the Kai proteins of the cyanobacterium Synechococcus elongatus PCC 7942 (S. e. PCC 7942), we set up the in vitro system to study the oscillation of KaiC's phosphorylation status based on SDS-PAGE. To study the structure-function relationship of KaiA, we designed different KaiA constructs. In this article, we presented our experimental data to further support our work in [1]. In Fig. 1, we presented the elution profiles of different KaiA constructs. In Fig. 2, we showed the SDS-PAGE gel images for testing the function of the wild-type KaiA (KaiAwt), the C-terminal domain of KaiA (KaiA-180C), and its concatenated form (KaiA-180Cd6).

Protein expression and purification
The expression and purification of KaiA, KaiB, and KaiC were similar with our previous report [2]. Briefly, all proteins were expressed as GST-tagged proteins, and then the GST tags were removed with PreScission Protease. The tag removed proteins were further purified with Hitrap FF Q columns. All protein coding sequences were verified with DNA sequencing.

Size exclusion chromatography
A Superdex 200 Increase 3.2/300 column (GE Healthcare) was used for evaluating the oligomerization states of the proteins. The proteins were prepared in the reconstitution buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM ATP, 5 mM MgCl 2 , and 0.01% Tween-20) and loaded to the column for analysis separately at room temperature with a flow rate of 0.01 mL/min.

in vitro reconstitution assay
The reconstitution assay was similar with our previous report [2]. Briefly, the purified proteins were incubated in the reaction buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM ATP, 5 mM MgCl 2 , and 0.01% Tween-20) at the ratio of KaiA:KaiB:KaiC ¼ 1:1:2 (m/v). The reaction system was incubated at 30°C and samples were collected at indicated time points. Finally, the samples were analyzed with 8% SDS-PAGE gels.

Evaluation of the SDS-PAGE gels
The SDS-PAGE gels were analyzed using Image J [4] with the gel analysis protocol as described in our published protocol as described in our published work [5].

Molecular dynamics simulation
The molecular dynamics simulation was performed in NAMD [6] as previous [2] using the KaiA homo-dimer structure in 5C5E (PDB ID). Periodic water boxes were added to wrap the protein with 10 Å of boundary distances. Na 2 þ and Cl − were added to 0.15 mol/L and counteracted the net chargers of the system. The Charmm parameters from c35b2_c36a2 were used, and the smooth particle-mesh Ewald (PME) method was enabled. The data were analyzed in VMD [7].

Acknowledgements
We would like to acknowledge the members of Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy, China Three Gorges University for supports.

Funding sources
This work was supported by the funds from the National Natural Science Foundation of China (21103098; 31670768), Hubei Province of China (D20161204), and China Three Gorges University.

Transparency document. Supplementary material
Transparency document associated with this article can be found in the online version at http://dx. doi.org/10.1016/j.dib.2018.03.032.