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dc.titleCyclic AMP- and (Rp)-cAMPS-induced conformational changes in a complex of the catalytic and regulatory (RIα) subunits of cyclic AMP-dependent protein kinase
dc.contributor.authorAnand, G.S.
dc.contributor.authorKrishnamurthy, S.
dc.contributor.authorBishnoi, T.
dc.contributor.authorKornev, A.
dc.contributor.authorTaylor, S.S.
dc.contributor.authorJohnson, D.A.
dc.identifier.citationAnand, G.S., Krishnamurthy, S., Bishnoi, T., Kornev, A., Taylor, S.S., Johnson, D.A. (2010-10). Cyclic AMP- and (Rp)-cAMPS-induced conformational changes in a complex of the catalytic and regulatory (RIα) subunits of cyclic AMP-dependent protein kinase. Molecular and Cellular Proteomics 9 (10) : 2225-2237. ScholarBank@NUS Repository.
dc.description.abstractWe took a discovery approach to explore the actions of cAMP and two of its analogs, one a cAMP mimic ((Sp)-adenosine cyclic 3′:5′- monophosphorothioate ((Sp)-cAMPS)) and the other a diastereoisomeric antagonist ((Rp)-cAMPS), on a model system of the type Iα cyclic AMP-dependent protein kinase holoenzyme, RIα(91-244)·C- subunit, by using fluorescence spectroscopy and amide H/2H exchange mass spectrometry. Specifically, for the fluorescence experiments, fluorescein maleimide was conjugated to three cysteine single residue substitution mutants, R92C, T104C, and R239C, of RIα(91-244), and the effects of cAMP, (S p)-cAMPS, and (Rp)-cAMPS on the kinetics of R-C binding and the time-resolved anisotropy of the reporter group at each conjugation site were measured. For the amide exchange experiments, ESI-TOF mass spectrometry with pepsin proteolytic fragmentation was used to assess the effects of (R p)-cAMPS on amide exchange of the RIα(91-244)·C-subunit complex. We found that cAMP and its mimic perturbed at least parts of the C-subunit interaction Sites 2 and 3 but probably not Site 1 via reduced interactions of the linker region and αC of RIα(91-244). Surprisingly, (Rp)-cAMPS not only increased the affinity of RIα(91-244) toward the C-subunit by 5-fold but also produced long range effects that propagated through both the C- and R-subunits to produce limited unfolding and/or enhanced conformational flexibility. This combination of effects is consistent with (Rp)-cAMPS acting by enhancing the internal entropy of the R·C complex. Finally, the (Rp)-cAMPS- induced increase in affinity of RIα(91-244) toward the C-subunit indicates that (Rp)-cAMPS is better described as an inverse agonist because it decreases the fractional dissociation of the cyclic AMP-dependent protein kinase holoenzyme and in turn its basal activity. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.
dc.contributor.departmentBIOLOGICAL SCIENCES
dc.description.sourcetitleMolecular and Cellular Proteomics
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