Resonant mirror biosensor analysis of type Ialpha cAMP-dependent protein kinase B domain--cyclic nucleotide interactions.
A resonant mirror biosensor was used to study cyclic nucleotide-receptor interactions. In particular, a novel method was developed to determine inhibition constants (Ki) from initial rates of ligate association to immobilized ligand. This approach was applied to the comparison of cyclic nucleotide-binding properties of the wild-type isolated B domain of the cAMP-dependent protein kinase type Ialpha regulatory subunit and its Ala-334-Thr (A334T) variant that has altered cyclic nucleotide specificity. A cUMP-saturated form of the B domain was used for all measurements. Under the conditions used, cUMP did not affect the kinetics of B domain association to immobilized cAMP. Triton X-100 was required to stabilize the protein at nanomolar concentrations. The association and dissociation rate constants for wild-type and A334T B domains yielded equilibrium dissociation constants of 11 and 16 nM. Heterogeneity of ligate and immobilized ligand, mass transport effects, and other factors were evaluated for their influence on biosensor-determined kinetic constants. Biosensor-determined relative inhibition constants (Ki' = Ki(cAMP)/Ki(analog)) for 16 cyclic nucleotide analogs correlated well with those determined by a [3H]cAMP binding assay. Previously published Ki' values for the B domain in the intact regulatory subunit were similar to those of the isolated B domain. The Ki' values for the wild-type and A334T B domains were essentially unchanged except for dramatic enhancements in affinity of cGMP analogs for the A334T B domain. These observations validate the isolated B domain as a simple model system for studying cyclic nucleotide-receptor interactions.
Muhonen
W W
WW
Department of Biochemistry and Molecular Biology, University of North Dakota School of Medicine, Grand Forks 58202-9037, USA.
Shabb
J B
JB
D019943:Amino Acid Substitution; D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics; D004347:Drug Interactions; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D009154:Mutation; D009712:Nucleotides, Cyclic / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D009712:Nucleotides, Cyclic; C517834:PRKAR1A protein, human; D017868:Cyclic AMP-Dependent Protein Kinases