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Structural and dynamic basis for the cAMP-mediated allosteric control of the catabolite activator protein (CAP)
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Title
Structural and dynamic basis for the cAMP-mediated allosteric control of the catabolite activator protein (CAP)
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ETD_1538
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http://hdl.rutgers.edu/1782.2/rucore10002600001.ETD.000051312
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eng
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theses
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Topic
Chemistry
Subject
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(authority = ETD-LCSH)
Topic
DNA-protein interactions
Subject
(ID = SBJ-3);
(authority = ETD-LCSH)
Topic
RNA polymerases
Subject
(ID = SBJ-4);
(authority = ETD-LCSH)
Topic
Transcription factors
Subject
(ID = SBJ-5);
(authority = ETD-LCSH)
Topic
Allosteric regulation
Abstract
The 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.
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.
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electronic resource
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xv, 164 p. : ill.
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Ph.D.
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Includes bibliographical references (p. 147-163)
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by Nataliya Popovych
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Popovych
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Nataliya
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Nataliya Popovych
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Kalodimos
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Charalampos G, Kalodimos
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Kalodimos
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Charalampos G. Kalodimos
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Jordan
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Frank
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Frank Jordan
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He
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Huixin
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Huixin He
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Ghose
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Ranajeet
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Ranajeet Ghose
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Rutgers University
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Graduate School - Newark
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2009
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2009-05
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xx
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Rutgers University Electronic Theses and Dissertations
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ETD
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Graduate School - Newark Electronic Theses and Dissertations
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rucore10002600001
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doi:10.7282/T3RX9C97
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ETD doctoral
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The author owns the copyright to this work.
Copyright
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Copyright protected
Notice
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Open
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Permission or license
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Popovych
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Nataliya
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Nataliya Popovych
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Rutgers University. Graduate School - Newark
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I hereby grant to the Rutgers University Libraries and to my school the non-exclusive right to archive, reproduce and distribute my thesis or dissertation, in whole or in part, and/or my abstract, in whole or in part, in and from an electronic format, subject to the release date subsequently stipulated in this submittal form and approved by my school. I represent and stipulate that the thesis or dissertation and its abstract are my original work, that they do not infringe or violate any rights of others, and that I make these grants as the sole owner of the rights to my thesis or dissertation and its abstract. I represent that I have obtained written permissions, when necessary, from the owner(s) of each third party copyrighted matter to be included in my thesis or dissertation and will supply copies of such upon request by my school. I acknowledge that RU ETD and my school will not distribute my thesis or dissertation or its abstract if, in their reasonable judgment, they believe all such rights have not been secured. I acknowledge that I retain ownership rights to the copyright of my work. I also retain the right to use all or part of this thesis or dissertation in future works, such as articles or books.
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