TY - JOUR
T1 - Amide H/2H exchange reveals communication between the cAMP and catalytic subunit-binding sites in the RIα subunit of protein kinase A
AU - Anand, Ganesh S.
AU - Hughes, Carrie A.
AU - Jones, John M.
AU - Taylor, Susan S.
AU - Komives, Elizabeth A.
N1 - Funding Information:
We thank Celina Juliano for assistance with protein purification. This work was supported in part by Howard Hughes Medical Institute, NIH grant GM34921 to S.S.T. and by NSF grant MCB9808286 to E.A.K. C.A.H. was supported by the Molecular Biophysics Training Program.
PY - 2002
Y1 - 2002
N2 - 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.
AB - 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.
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U2 - 10.1016/S0022-2836(02)00919-1
DO - 10.1016/S0022-2836(02)00919-1
M3 - Article
C2 - 12381327
AN - SCOPUS:0036025308
VL - 323
SP - 377
EP - 386
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
SN - 0022-2836
IS - 2
ER -