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

AbstractCharacterization of the unfolding dynamics of a recombinant type IA regulatory subunit (RIα) of cyclic adenosine monophosphate (cAMP)‐dependent protein kinase (cAPK) was examined by CE with UV detection. Electrophoretic separation of RIα 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 ligand relative to receptor. This process enabled in‐capillary generation of cAMP‐stripped RIα, which was used to estimate the apparent dissociation constant (Kd) of 0.6 ± 0.2 μM. A comparison of RIα dynamic unfolding processes with urea denaturation was performed by CE with (i.e., RIα–cAMP) and without (i.e., cAMP‐stripped RIα) 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 (ΔGU°) of 10.1 ± 0.5 kcal·mol+1 and greater cooperativity in unfolding (m) of −2.30 ± 0.11 kcal·mol−1M−1. CE offers a rapid, yet versatile platform for probing the thermodynamics of cAPK and other types of receptor–ligand complexes in free solution.

Dynamic unfolding of a regulatory subunit of cAMP‐dependent protein kinase by capillary electrophoresis: Impact of cAMP dissociation on protein stability Journal Articles