{'fraction_non_alphanumeric': 0.025266704098820886, 'fraction_numerical': 0.01403705783267827, 'mean_word_length': 5.674157303370786, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Resonant mirror biosensor analysis of type Iα 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 Iα 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 (K'i = KicAMP/K'ianalog) for 16 cyclic nucleotide analogs correlated well with those determined by a [3H]cAMP binding assay. Previously published K'i values for the B domain in the intact regulatory subunit were similar to those of the isolated B domain. The K'i 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.