Dynamic unfolding of a regulatory subunit of cAMP-dependent protein kinase by capillary electrophoresis: Impact of cAMP dissociation on protein stability.

Characterization of the unfolding dynamics of a recombinant type IA regulatory subunit (RIalpha) of cyclic adenosine monophosphate (cAMP)-dependent protein kinase (cAPK) was examined by CE with UV detection. Electrophoretic separation of RIalpha by CE in a buffer devoid of cAMP resulted in rapid dissociation of the complex from the original sample due to the high negative mobility of the ligandrelative to receptor. This process enabled in-capillary generation of cAMP-stripped RIalpha, which was used to estimate the apparent dissociation constant (Kd) of 0.6 +/- 0.2 microM. A comparison of RIalpha dynamic unfolding processes with urea denaturation was performed by CE with (i.e., RIalpha-cAMP) and without (i.e., cAMP-stripped RIalpha) excess cAMP in the buffer during electromigration. The presence of cAMP in the buffer confirmed greater stabilization of the protein, as reflected by a higher standard free energy change (DeltaG(U) degrees) of 10.1 +/- 0.5 kcal x mol(+1) and greater cooperativity in unfolding (m) of -2.30 +/- 0.11 kcal x mol(-1) M(-1). CE offersa rapid, yet versatile platform for probing the thermodynamics of cAPK and othertypes of receptor-ligand complexes in free solution.