Journal
BIOCHEMISTRY
Volume 39, Issue 50, Pages 15626-15632Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi002196l
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- NIGMS NIH HHS [GM 34921] Funding Source: Medline
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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-alpha(I)) 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-alpha(I), 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.
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