The changes in backbone hydrogen/deuterium (H/2H) exchange in the regulatory subunit (RIα(94-244)) of cyclic AMP-dependent protein kinase A (PKA) were probed by MALDI-TOF mass spectrometry. The three naturally occurring states of the regulatory subunit were studied: (1) free RIα(94-244), which likely represents newly synthesized protein, (2) RIα(94-244) bound to the catalytic (C) subunit, or holoenzyme, and (3) RIα(94-244) bound to cAMP. Protection from amide exchange upon C-subunit binding was observed for the helical subdomain, including the A-helix and B-helix, pointing to regions adjacent to those shown to be important by mutagenesis. In addition, C-subunit binding caused changes in observed amide exchange in the distal cAMP-binding pocket. Conversely, cAMP binding caused protection in the cAMP-binding pocket and increased exchange in the helical subdomain. These results suggest that the mutually exclusive binding of either cAMP or C-subunit is controlled by binding at one site transmitting long distance changes to the other site.
All Science Journal Classification (ASJC) codes
- Structural Biology
- Molecular Biology