Abstract
The slow but temperature-insensitive adenosine triphosphate (ATP) hydrolysis reaction in KaiC is considered as one of the factors determining the temperature-compensated period length of the cyanobacterial circadian clock system. Structural units responsible for this low but temperature-compensated ATPase have remained unclear. Although whole-KaiC scanning mutagenesis can be a promising experimental strategy, producing KaiC mutants and assaying those ATPase activities consume considerable time and effort. To overcome these bottlenecks for in vitro screening, we optimized protocols for expressing and purifying the KaiC mutants and then designed a high-performance liquid chromatography system equipped with a multi-channel high-precision temperature controller to assay the ATPase activity of multiple KaiC mutants simultaneously at different temperatures. Through the present protocol, the time required for one KaiC mutant is reduced by approximately 80% (six-fold throughput) relative to the conventional protocol with reasonable reproducibility. For validation purposes, we picked up three representatives from 86 alanine-scanning KaiC mutants preliminarily investigated thus far and characterized those clock functions in detail.
Highlights
Circadian clocks are self-sustained oscillatory systems allowing organisms to anticipate and adapt to the daily environmental changes resulting from the Earth’s rotation [1]
Circadian rhythms arising as consequences of negative feedback regulations of clock gene transcriptions by the clock proteins are called transcriptional and translational oscillations (TTO) [5], while those driven solely by the clock proteins are called post-translational oscillations (PTO) [6]
KaiC ATPase is of particular interest here, as its activity is extremely low (~12 adenosine triphosphate (ATP) d-1) [10], temperature compensated (Q10 = 1.0) [10], and finely correlated to the frequencies of TTO as well as PTO [10,13]
Summary
Circadian clocks are self-sustained oscillatory systems allowing organisms to anticipate and adapt to the daily environmental changes resulting from the Earth’s rotation [1]. KaiC ATPase is of particular interest here, as its activity is extremely low (~12 ATP d-1) [10], temperature compensated (Q10 = 1.0) [10], and finely correlated to the frequencies of TTO as well as PTO [10,13] These unique properties inspire development of an ATPase-based screening for KaiC clock mutants giving short, long, and/or temperature-dependent periods, as one of the effective methods to uncover origins of the temperature-compensated circadian period in the S. elongatus clock system. A new high-performance liquid chromatography (HPLC) system with a four-channel temperature controller was designed to determine temperature dependencies of the ATPase activities for multiple KaiC mutants simultaneously These improvements together with several other optimizations reduced approximately 80% of the time costs associated with the overall screening process. We discuss future perspectives of the ATPase-based in vitro screening on the basis of advantages and disadvantages of the present method
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