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Structural and dynamic basis for the cAMP-mediated allosteric control of the catabolite activator protein (CAP)
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Popovych, Nataliya. Structural and dynamic basis for the cAMP-mediated allosteric control of the catabolite activator protein (CAP). Retrieved from https://doi.org/doi:10.7282/T3RX9C97

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TitleStructural and dynamic basis for the cAMP-mediated allosteric control of the catabolite activator protein (CAP)
NamePopovych, Nataliya (author); Kalodimos, Charalampos (chair); Kalodimos, Charalampos (chair); Jordan, Frank (internal member); He, Huixin (internal member); Ghose, Ranajeet (outside member); Rutgers University; Graduate School - Newark
Date Created
Other Date2009-05 (degree)
SubjectChemistry, DNA-protein interactions, RNA polymerases, Transcription factors, Allosteric regulation
Extentxv, 164 p. : ill.
DescriptionThe E.coli catabolite activator protein (CAP) is a universal transcriptional activator that regulates more than half of the cell’s transcription units by responding to fluctuations of the cellular concentration of cAMP. Cyclic 3’-5’-adenosine monophosphate (cAMP) binding to CAP elicits an allosteric transition that switches CAP from the “off” state, which binds DNA weakly and nonspecifically, to the “on” state that binds DNA strongly and specifically. In the cAMP-bound state CAP binds to DNA sites located at or close to promoter sites and enhances RNA polymerase binding to the promoter by a simple recruitment mechanism thereby activating transcription.
The solution structure of CAP in the absence of cAMP (apo-CAP) was determined by nuclear magnetic resonance (NMR) spectroscopy. Multidimensional heteronuclear NMR experiments in combination with a series of specifically labeled samples were used to accomplish structure determination.
The structure of the 50-kDa apo-CAP provides atomic details of the allosteric transition that leads to activation of CAP for DNA binding. cAMP elicits a coil-to-helix transition that stabilizes a suboptimal coiled coil, which is extensively unwound in apo-CAP. Coiled-coil winding causes the DNA-binding domain to undergo a large rigid-body translation and rotation that poise the recognition helices for specific DNA binding. The results highlight the unique structural mechanisms that underlie activation of this prototypic transcription factor and provide an example of how effector-mediated structural changes can control the activity of regulatory proteins. In addition, we have also characterized a number of CAP* mutants in order to further understand the mechanism underlying the activation of CAP.
NMR spectroscopy in combination with isothermal titration calorimetry (ITC) was used to delineate the structural and dynamic features of distinct conformational states in cAMP binding to CAP. The intrinsically cooperative binding of cAMP to CAP was characterized using the isolated effector binding domain CAPN (residues 1-138) in three conformational states: in the absence of cAMP (apo-CAPN), in singly liganded state (intermediate, cAMP1-CAPN), and in the doubly liganded state (cAMP2-CAPN). We have shown that allosteric interactions can be mediated exclusively by transmitted changes in protein motions. Thus, the observed protein allostery is exclusively dynamically driven.
NotePh.D.
NoteIncludes bibliographical references (p. 147-163)
Noteby Nataliya Popovych
Genretheses, ETD doctoral
Persistent URLhttps://doi.org/doi:10.7282/T3RX9C97
Languageeng
CollectionGraduate School - Newark Electronic Theses and Dissertations
Organization NameRutgers, The State University of New Jersey
RightsThe author owns the copyright to this work.
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