Protein Systems for Measuring 24 Hours - Basic Design of a Circadian Clock
Nagoya University, Graduate School of Science
Professor Emeritus, Takao Kondo
Lives on Earth live skillfully by adapting to a 24-hour day/night cycle. We, human beings,
carry wristwatches and use them in our daily lives, but animals, plants, and bacteria that do nothave wristwatches also have clocks in their cells that program their daily lives.

This clock, calleda circadian clock (biological clock), is so accurate that it hardly deviates even if the temperaturechanges. This is very strange, considering that temperature greatly affects biological activity.
Of course, we also have a circadian clock that precisely regulates various bodily functions and sleep.
The question of how life remembers the Earth's rotation period in its cells and generates
temperature-independent oscillations over 24 hours has fascinated researchers in a wide range offields.

In 1992, we developed an experimental system in cyanobacteria using bioluminescenceand found three clock genes, kaiA, kaiB, and kaiC (kai named after rotation), and showed that theexpression of kaiC was similar to the clock model based on gene expression proposed in higherorganisms. However, we gradually came to believe that this model could not explain the stable
24-hour period length and sought to elucidate the biochemical function of the KaiC protein, which
was thought to play a key role in solving this mystery.

As a result, in 2005, we discovered that atemperature-independent 24-hour rhythm is generated just by mixing three Kai proteins and ATP
in a test tube and succeeded in reconstructing the circadian clock in vitro. (Figure 1). Surprisingly,the rhythm of proteins are very stable, and the period is temperature-compensated, and it has thesame properties as the circadian clock of living cells and individuals.

This discovery was a

Copernican turn for studying circadian clocks, which were thought to be a function of living cells,
and had an enormous impact on investigating clocks in higher organisms. This achievement has
also attracted the attention of researchers in the fields of chemistry and physics since it revealed
an entirely new function of proteins in "ticking time.

HourFig. 1. Circadian Clock by ProteinKaiC repeats phosphorylation autonomously in vitro in an approximately 24-hour cycle. Purified
KaiA and KaiC were mixed in vitro with ATP and kept at 30°C. A small amount was taken every
2 hours to stop the reaction, and the state of KaiC was examined by electrophoresis; the KaiC
protein was detected in two bands, the upper phosphorylated form, and the lower
dephosphorylated form. The figure clearly shows that the phosphorylation state oscillated in a24-hour cycle. This period length was temperature compensated, and the mutant protein
exhibited the same period as rhythm of the mutant cell.

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How does the Kai protein count 24 hours? The leading player, KaiC, is composed of two
ATP-degrading enzymes (ATPases); ATP is the "currency of the cell" and is the direct energy
source molecule for most biological activities. Life uses various ATPases to break down ATP toobtain energy for vital activities. When we measured the activity of KaiC quantitatively, we once
again surprised by the results (Fig. 2).

First of all, the activity was extremely low, degrading only

10-15 ATP per day. This activity is so low (almost zero level) that it seems to be an enzyme thathas lost its function. Still, upon closer examination, this activity is not affected by temperatureand is highly stable. When further examined in a mutant KaiC with an altered period, this activity
was proportional to the speed (frequency) of the circadian clock. This means that the activity of
the ATPase determines the characteristics of the circadian clock (period length and its
temperature conpensation). More importantly, this characteristic is found even in KaiC incubatedalone, which does not generate rhythms. This indicates that KaiC does measure period length
by generating rhythms but rather memorizes 24 hours as an intrinsic property within KaiCstructure.

ATPase activity

ATPase activity

day

Number of ATP hydrolysis

Temperature(per day/KaiC)

Figure 2. ATPase activity of KaiC. Only KaiC
was placed with ATP, and ATPase activity wasmeasured. No phosphorylation rhythm occurredin the case of KaiC alone. (A) The ATPase
activity of KaiC is very low but precisely
temperature compensated. (B) Furthermore,this activity is proportional to the frequency(speed) of the circadian clock. The ATPase
activity of the wild type and the five periodic
mutants is plotted against their frequency(reciprocal of the number of cycles).

How does KaiC's ATPase realize this?

This is the most fundamental question of thecircadian clock, yet it is not well understood.

However, with the amount of energy used, it is

Mechanical Pendulum
Clock

KaiC Protein

impossible to explain the 24-hour oscillation withthe sequential combination of chemical reactions,and a completely different mechanism seems tobe required.

Recent studies have examined thepossibility that the ATP-degradation energy
from CI-ATPase causes distortions in its
molecular structure and mechanically suppresses
its own ATP-degradation activity. This process
would
Figure 3: Mechanical Pendulum Clock andKaiC Protein Designs
Both are composed of very similar designs,
although the entities are quite different. See
text for details.

establish

stableregulation (Figure 3).

negative

feedback

If this is realized, aspring-like mechanism would be maintained in
the internal strain of the protein, and it will bepossible to function as a simple harmonicCopyright © Ohsumi Frontier Science Foundation. All Rights Reserved.

oscillator which period is independent of temperature or amplitude, like a balance in a mechanicalwristwatch. However, harmonic oscillation of CI alone would, sooner or later, stop anyway andwould not be able to transmit period to outside.
Therefore, it is important that another ATPase (CII) is used to couple it with a pacemaker(CI-ATPase) to sustain CI oscillations. KaiC's CIIATPase collaborates with KaiA and KaiB tosequentially repeat phosphorylation and dephosphorylation of two adjacent amino acids of CII.
The cycle of this process is naturally affected by temperature and various environmental factors.
But if the pacemaker of CI gates its progression, it will cycle as CI-oscillation. And suppose CI
can receive a very small amount of mechanical energy from CII somewhere in this cycle.

In thatcase, CI can acquire energy in the same cycle that could sustain the CI oscillation. In this way,
the two cycles can continue to stable oscillations of the same period, and CII can also obtain astable period and control various metabolic activities in a 24-hour cycle. In other words, it has
combined two different types of oscillations with different properties and has a good combinationof the two. In fact, this configuration is similar to the design of mechanical watches, and thecoupling mechanism is called an escapement mechanism.

The escapement connects theisochronous pendulum discovered by Galileo to a rotating tool with a faster periodic spring orweight. The mechanism developed by Huygens, Hooke, and many subsequent watchmakers
restricts the direction of action to one direction and the duration of action to a very short period,
allowing the pacemaker (pendulum or balance) is designed to continue free harmonic oscillation
by reliably controlling the spring-driven rotating mechanism while ensuring that very little energy
is gained.
Is such a mechanism possible within KaiC?

It will take some time to reveal the atomic

structure, since the movements will likely be a slight movement at the atomic level. But among
many mutants of KaiC isolated, some mutants showed the abnormalities in this function.

In thismutant, the cycle is affected by temperature, and the cycle becomes 8-10 hours, and thephosphorylation cycle seems to rotate freely, with no temporal resting period as found in the wildtype.

The presence of these mutants expects to have a clock mechanism similar to that of

mechanical clocks in proteins.

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