Please use this identifier to cite or link to this item:
|Title:||Cooperativity and allostery in cAMP-dependent activation of Protein Kinase A: Monitoring conformations of intermediates by amide hydrogen/deuterium exchange|
|Citation:||Moorthy, B.S., Badireddy, S., Anand, G.S. (2011-04-30). Cooperativity and allostery in cAMP-dependent activation of Protein Kinase A: Monitoring conformations of intermediates by amide hydrogen/deuterium exchange. International Journal of Mass Spectrometry 302 (1-3) : 157-166. ScholarBank@NUS Repository. https://doi.org/10.1016/j.ijms.2010.09.012|
|Abstract:||Amide hydrogen/deuterium exchange mass spectrometry is a powerful method both for mapping protein-protein interactions and measuring conformational dynamics of protein complexes. In this study we report its application to monitoring the stepwise process governing cAMP-dependent activation of Protein Kinase A (PKA). In the absence of cAMP, PKA exists in an inactive complex of catalytic (C) and regulatory (R) subunits. cAMP binding induces large conformational changes within the R-subunit leading to dissociation of the active C-subunit. Although crystal structures of end-point, inactive and active states are available, the molecular basis for cooperativity in cAMP-dependent activation of PKA is not clear. In this study we report application of amide hydrogen/deuterium exchange mass spectrometry on tracking the stepwise cAMP-induced conformational changes using a single point mutant (R209K) at the cyclic nucleotide binding domain (CNB)-A site. Our amide exchange results reveal that binding of one molecule of cAMP increases amide exchange in important regions within the second CNB-B domain. Increased exchange was also seen at the interface between CNB-B and the C-subunit suggesting weakening of the R-C interface without dissociation. Importantly, binding of the first molecule of cAMP greatly increases the conformational mobility/dynamics of two key regions coupling the two CNBs, the αC/C′:A and αA:B helix. We believe that the enhanced dynamics of these regions forms the basis for the positive cooperativity in the cAMP-dependent activation of PKA. In summary, our results reveal the close allosteric coupling between CNB-A and CNB-B with the C-subunit providing important molecular insights into the function of CNB-B domain. © 2010 Elsevier B.V.|
|Source Title:||International Journal of Mass Spectrometry|
|Appears in Collections:||Staff Publications|
Show full item record
Files in This Item:
There are no files associated with this item.
checked on Jan 24, 2019
WEB OF SCIENCETM
checked on Jan 16, 2019
checked on Jan 18, 2019
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.