title: Consequences of cAMP and catalytic-subunit binding on the flexibility of the A-kinase regulatory subunit.
abstract: A combination of site-directed labeling and time-resolved fluorescence anisotropy was used to further elucidate the structure and underlying dynamic features of the type I regulatory (R(I)(alpha)) subunit of the cAMP-dependent protein kinase. Specifically, the consequences of cAMP and the catalytic (C)-subunit binding on the backbone flexibility around seven sites of cysteine substitution and fluorescein maleimide labeling (Thr(6)Cys, Leu(66)Cys, Ser(75)Cys, Ser(81)Cys, Ser(99)Cys, Ser(145)Cys, and Ser(373)Cys) in the R(I)(alpha) subunit were assessed. Focusing on the fast rotational correlation time, the results indicate that most of the interdomain segment connecting the dimerization/docking (D/D) and tandem cAMP-binding domains is probably weakly associated with the latter domain. Also, this segment becomes more tightly bound to the C subunit upon holoenzyme formation. The results also suggest that there is a short 'hinge' segment (around Leu(66)Cys) that could allow the structured interdomain/cAMP-binding and D/D domains to pivot about each other. Finally, cAMP binding dramatically reduces the backbone flexibility around only the two sites of cysteine substitution in the cAMP-binding domains, suggesting a selective structural stabilization caused by cAMP and a "tight" coupling of low-nanosecond fluctuations selectively within the tandem cAMP-binding domains.
authors: Division of Biomedical Sciences, University of California, Riverside, California 92521-0121, USA.
authors: F
authors: F
authors: Li
authors: M
authors: M
authors: Gangal
authors: J M
authors: JM
authors: Jones
authors: J
authors: J
authors: Deich
authors: K E
authors: KE
authors: Lovett
authors: S S
authors: SS
authors: Taylor
authors: D A
authors: DA
authors: Johnson
mesh_terms: D002384:Catalysis; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation; D009154:Mutation; D011487:Protein Conformation; D013329:Structure-Activity Relationship
chemical_list: D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases