The catalytic (C) and regulatory (R) subunits of Protein Kinase A (PKA) are exceptionally dynamic proteins. Interactions between the R- and C-subunits are regulated by cAMP binding to the two cyclic nucleotide-binding domains in the R-subunit. Mammalian cells express four different isoforms of the R-subunit (RIalpha, RIbeta, RIIalpha, and RIIbeta) that all interact with the C-subunit in different ways. Here, we investigate the dynamic behavior of protein complexes between RIalpha and C-subunits using small angle X-ray scattering. We show that a single point mutation in RIalpha, R333K (which alters the cAMP-binding properties of Domain B) results in a compact shape compared to the extended shape of the wild-type R:C complex. A double mutant complex that disrupts the interaction site between the C-subunit and Domain B in RIalpha, RIalpha(AB)R333K:C(K285P), results in a broader P(r) curve that more closely resembles the P(r) profiles of wild-type complexes. These results together suggest that interactions between RIalpha Domain B and the C-subunit in the RIalpha:C complex involve large-scale dynamics that can be disrupted by single point mutations in both proteins. In contrast to RIalpha:C interactions, Domain B in the RIIbeta:C heterodimer is not dynamic and is critical for both inhibition and complex formation. Our study highlights the functional differences of domain dynamics between PKA isoforms, providing a framework for elucidating the global organization of each holoenzyme and the crosstalk between the R- and C-subunits.