Direct measurements of clock-protein dynamics using quasielastic neutron scattering
CIMoS, Institute for Molecular Science, NINS

Shuji AKIYAMA

1. Introduction
Circadian (approximately 24 h) clocks are endogenous timing systems enabling organisms to fit dailychanges in environments. These time-measuring systems share three properties. First, the clock systems reveal anoscillation with a circadian period even without any external cues (self-sustained oscillation). Second, the period of
the clock systems is little dependent on the temperature (temperature compensation). Third, the phase of theoscillator can be shifted forward/backward in response to the changes of light/temperature to effect the fitness tothe diurnal fluctuation of the environment (synchronization).
It has become widely accepted, especially in eukaryotes, that clock genes and their translational products(clock proteins) constitute a negative-feedback loop as a core oscillator to realize transcriptional-translational
oscillation (TTO). On the other hand, both the transcription and translation proceed in the order of minutes, and thus
it remains unclear how and why the circadian systems composed of fast-moving macromolecules can be so slowand stable.
To address the issue, we have used cyanobacterium Synechococcus elongatus PCC 7942 as the model
system and looked for the minimal-and-slow reaction correlated to the frequency of the clock oscillator (Abe et al.
Science 2015). The rate-limiting reaction we discovered was the steady-state ATPase activity (11 d-1) in the Nterminal half of the clock protein KaiC (KaiC1). The ATPase activity in KaiC1 behaves as the pacemaker of in vivo
frequency of TTO rhythm; i.e. the higher ATPase in vitro resulted in higher frequency of TTO in vivo. Furthermore,
we recorded the time-response curves of KaiC ATPase, and our system-control analyses uncovered a naturalfrequency of the KaiC ATPase. Astonishingly, the natural frequency of KaiC ATPase was 0.91 d-1, in good agreementwith the frequency of the in vivo cyanobacterial clock (1 d-1). This is the first experimental demonstration that invivo frequency of TTO is related on one-to-one correspondence to the rate of minimal biochemical reaction within
the particular clock protein. This correspondence implies that the rotation period of the Earth is inherently “encoded”
behind the regulatory mechanism of slow KaiC ATPase.
In this research proposal, we conducted a series of quasi-elastic neutron scattering (QENS) experiments
to address following two points;
1. Correlation between KaiC dynamics and ATPase activity (circadian clock frequency)
2. Mechanism behind temperature compensation of KaiC ATPase.

2. ExperimentWild-type KaiC (KaiC-WT), a short-period KaiC mutant (KaiC-SP), and a long-period KaiC mutant(KaiC-LP) were expressed in E. coli. and purified as described previously (EMBO J. 26, 4029-4037, 2007). After abuffer exchange into a D2O buffer, the KaiC samples were concentrated up to approximately 10 mg/ml using
ultrafiltration. QENS measurements were conducted for the KaiC samples and the D2O buffer at five different
temperatures between 278 and 313 K using the near-backscattering spectrometer, BL02 (DNA), at J-PARC MLF.
The obtained QENS spectra, S(Q,ω), were corrected for contributions from an empty cell and for detector efficiency,and then normalized relative to that of a vanadium standard.

1Figure 1. QENS profiles at 313 K for a sample solution of KaiC-WT (a), a D2O buffer (b), and KaiC-WT after
subtracting contributions of D2O (c).

3. Results
Figs. 1a and 1b show QENS profiles for the KaiC sample and the D2O buffer, respectively, recorded at313 K. Contributions from the D2O buffer (Fig. 1b) were subtracted from the sample dataset (Fig. 1a) using scaling
factors calculated from theoretical scattering cross section, and then a resultant QENS spectrum (Fig. 1c) of wildtype KaiC (KaiC-WT) was fitted to the following equation,
S,,

⨂,

⨂,

where A0(Q) and A1(Q) are amplitudes of global and local motions, respectively;
δ(ω) is Dirac delta-function; Llocal(Q,ω) and Lglobal(Q,ω) are Lorentzianfunctions describing local and global atomic motions, respectively; R(Q,ω) is
an instrumental resolution function; BG(Q) is a background function. Fig.2 is atypical result of fitting to the experimental QENS spectrum of KaiC-WT at Q =1.225 Å-1.
A half-width at half-maximum (HWHM) of Lglobal(Q,ω), Γglobal(Q),provides information on diffusive motions of proteins. The Γglobal(Q) values forKaiC-WT increased linearly with Q2. Apparent diffusion coefficients, Dapp,were estimated from the linear slope of a plot of Γglobal(Q) against Q2. The Dappvalues of KaiC-SP (4.42 ± 0.22 × 10-7 cm2 s-1) and KaiC-LP (4.30 ± 0.17 × 10- Figure 2. QENS spectrum of KaiC-WT
at Q = 1.225 Å−1 at 313 K.

7cm2 s-1) were indistinguishable from that of KaiC-WT (4.42 ± 0.14 × 10-7cm2 s-1) at 313 K, indicating that all KaiC samples adopted the functional hexamer throughout the QENSmeasurements.
A HWHM of Llocal(Q,ω), Γlocal (Q), provides information on local motions of proteins. Q2-dependency ofΓlocal(Q) was analyzed under the assumption of jump-diffusion model to estimate local residence time (τ).
Interestingly, the τ value for KaiC-WT revealed a unique temperature dependence. A slope of an Arrhenius plot ofthe τ value for KaiC-WT is seemingly different from each of other two KaiC mutants, suggesting a potentialcorrelation between the speed of the clock oscillator (ATPase activity) and the local motions in KaiC.

4. Conclusion
Although great care must be taken on interpreting the present data with a limited S/N ratio, the local
motions in KaiC are seemingly more sensitive to the clock property than we expected. We believe that our first
QENS experiment conducted under the approval of the trial use is successful and should be a good starting point tofurther experiments using a variety of the KaiC mutants.
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