Biyolojik saat proteinleri arasındaki etkileşimin yüzey plasmon rezonans tekniği ile incelenmesi

Circadian clock is an internal time keeping system recurring 24 hour 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 E-box 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 (SPR). The pair wise 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 SPR data with pseudo first order reaction kinetics approximation, and via nonlinear regression curve fitting. The results indicated that CRY2, PER2 and BMAL1, CLOCK proteins form complexes in vitro, and that PER2, CRY2 and PER2:CRY2 complex has similar affinities towards BMAL1:CLOCK complex. CRY2 protein had the highest affinity toward EBOX complex while 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, to address the behavioral and physiological disorders, and will be useful for the design of new drugs towards clock related diseases.