Here we present the use of PBD to determine the kinetic and thermodynamic parameters associated with calcium binding to DREAM protein. The fast (10 �s) release of calcium was photo triggered using the photosensitive calcium chelator DM-nitrophen (DMNP). The PBD traces for photolysis of Ca2+DMNP show a fast 300 �s decay, whereas upon addition of DREAM(65-256) an additional slow phase with  a decay of  1 ms can be resolved. Moreover, a construct lacking the first 161 amino acids shows a slower 2 ms decay. The differences in kinetics can be correlated with the oligomerization transition associated with each construct, where DREAM(65-256) undergoes a tetramer to dimer and DREAM(161-256) a monomer to dimer transition upon calcium binding. Furthermore, the slow transition is measured to be exothermic with a concomitant volume expansion. Together, these results establish the implementation of PBD for dynamic and thermodynamic study of calcium induced transitions in DREAM protein.

Study of the dynamic and thermodynamic calcium induced transition in the downstream regulatory element antagonist modulator (DREAM) using photothermal beam deflection (PBD)

245th National Meeting (2013)

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