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 ligand relative 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 offers a rapid, yet versatile platform for probing the thermodynamics of cAPK and other types of receptor-ligand complexes in free solution.
Gavina
Jennilee M A
JM
Department of Chemistry, McMaster University, Hamilton, Canada.
Das
Rahul
R
Britz-McKibbin
Philip
P
D000242:Cyclic AMP / Q000737:chemistry; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry*; D004213:Dissociative Disorders; D019075:Electrophoresis, Capillary / Q000379:methods*; D004795:Enzyme Stability; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D017510:Protein Folding; D021122:Protein Subunits / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry
D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D008024:Ligands; C517834:PRKAR1A protein, human; D021122:Protein Subunits; D011994:Recombinant Proteins; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases