Quantification of interactions among circadian clock proteins via surface plasmon resonance

Abstract

Circadian clock is an internal time keeping system recurring 24 h daily rhythm in physiology and behavior of organisms. Circadian clock contains transcription and translation feedback loop involving CLOCK/NPAS2, BMAL1, Cry1/2, and Per1/2. In common, heterodimer of CLOCK/NPAS2 and BMAL1 binds to EBOX element in the promoter of Per and Cry genes in order to activate their transcription. CRY and PER making heterodimeric complexes enter the nucleus in order to inhibit their own BMAL1-CLOCK-activated transcription. The aim of this study was to investigate and quantify real-time binding affinities of clock proteins among each other on and off DNA modes using surface plasmon resonance. The pairwise interaction coefficients among clock proteins, as well as interaction of PER2, CRY2, and PER2 : CRY2 proteins with BMAL1 : CLOCK complex in the presence and absence of EBOX motif have been investigated via analysis of surface plasmon resonance data with pseudo first-order reaction kinetics approximation and via nonlinear regression curve fitting. The results indicated that CRY2 and PER2, BMAL1, and CLOCK proteins form complexes in vitro and that PER2, CRY2 and PER2 : CRY2 complex have similar affinities toward BMAL1 : CLOCK complex. CRY2 protein had the highest affinity toward EBOX complex, whereas PER2 and CRY2 : PER2 complexes displayed low affinity toward EBOX complex. The quantification of the interaction between clock proteins is critical to understand the operation mechanism of the biological clock and to address the behavioral and physiological disorders, and it will be useful for the design of new drugs toward clock-related diseases.

Combined multiple transcriptional repression mechanisms generate ultrasensitivity and oscillations

“…The blocking-and sequestration-type repressions also effectively occur in the mammalian circadian clock. That is, PER:CRY binds with CLOCK:BMAL1 tightly (i.e., small 𝐾 𝑠 ), and CRY binds with CLOCK:BMAL1:E-box tightly (i.e., small 𝐾 𝑏 ) [45]. Such tight bindings are important for strong rhythm generation (figure 4e, black solid line) according to our model prediction (figure 3e).…”

Mechanistic insight into light-dependent recognition of Timeless by Drosophila Cryptochrome

“…Photoactive proteins, both natural and designed, typically change affinity for their targets by at least an order of magnitude when in light-activated states (Akiyama et al, 2016;Kepsutlu et al, 2014;Kondoh and Terazima, 2017;Zimmerman et al, 2016). Whereas a dark-state affinity in the $30 mM range for CRY is likely sufficient to arrest TIM degradation, an increase in affinity for TIM by only $3.5-fold for the WT protein seems modest.…”

Combined multiple transcriptional repression mechanisms generate ultrasensitivity and oscillations

“…small ), and CRY binds with CLOCK:BMAL1:E-box tightly (i.e. small ) [ 46 ]. Such tight bindings are important for strong rhythm generation ( figure 4 e , black solid line) according to our model prediction ( figure 3 e ).…”