title: 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.
authors.affiliation: Chemical and Biological Engineering, Koc University, Sariyer, Istanbul, 34450, Turkey.
authors.forename: Burcu
authors.initials: B
authors.lastname: Kepsutlu
authors.forename: Riza
authors.initials: R
authors.lastname: Kizilel
authors.forename: Seda
authors.initials: S
authors.lastname: Kizilel
mesh_terms: D056930:ARNTL Transcription Factors / Q000235:genetics / Q000378:metabolism; D000818:Animals; D056926:CLOCK Proteins / Q000235:genetics / Q000378:metabolism*; D057906:Circadian Clocks / Q000235:genetics*; D056931:Cryptochromes / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D056950:Period Circadian Proteins / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D025941:Protein Interaction Mapping
chemical_list: D056930:ARNTL Transcription Factors; C538960:BMAL1 protein, human; C538966:CRY2 protein, human; D056931:Cryptochromes; C421902:PER2 protein, human; D056950:Period Circadian Proteins; D056926:CLOCK Proteins
keywords: EBOX; circadian rhythm; clock proteins; dissociation constant; nonlinear regression curve fitting; surface plasmon resonance (SPR)