Kinetic Folding Studies of Apaf-1 CARD and Procaspase-3

Show simple item record

dc.contributor.advisor Carol Hall, Committee Member en_US
dc.contributor.advisor William Miller, Committee Member en_US
dc.contributor.advisor A. Clay Clark, Committee Chair en_US
dc.contributor.advisor Michael Goshe, Committee Member en_US
dc.contributor.author Milam, Sara Lis en_US
dc.date.accessioned 2010-04-02T18:41:33Z
dc.date.available 2010-04-02T18:41:33Z
dc.date.issued 2008-11-13 en_US
dc.identifier.other etd-10292008-113043 en_US
dc.identifier.uri http://www.lib.ncsu.edu/resolver/1840.16/3984
dc.description.abstract Apoptosis regulates the balance between cell growth and death. Apoptosis consists of two main signaling pathways, extrinsic and intrinsic. We have examined the kinetic folding mechanism of two proteins, Apaf-1 CARD and procaspase-3, which play a major part in these signaling cascades. The caspase recruitment domain (CARD) of Apaf-1 (apoptotic protease activating factor) is the 97 amino acid N-terminal domain involved in protein-protein interactions, allowing for the activation of procaspase-9. Apaf-1 CARD consists of six antiparallel α-helices arranged in a Greek key topology. Single and sequential mixing stopped-flow studies showed that Apaf-1 CARD folds and unfolds rapidly and suggest a folding mechanism that contains parallel channels with two unfolded conformations folding to the native conformation. KINSIM simulations show that a slow folding phase is described by a third conformation in the unfolded ensemble that interconverts with one or both unfolded species. Overall, the native ensemble is formed rapidly upon refolding. Procaspase-3, like all other caspases, exists in the cell as an inactive zymogen and is the final step in the apoptotic pathway. Once activated it cleaves numerous substrates, leading to the dismantling of the cell. The refolding pathway of homodimeric procaspase-3 is complex, consisting of multiple monomeric intermediates with a slow rate of dimerization. The refolding and unfolding burst phase revealed multiple species formed within milliseconds of folding. Kinetic data support the hypothesis of two native conformations, one of which is enzymatically active. Collectively, these results demonstrate that dimerization is an important aspect in both folding and activation of procaspase-3. Overall, the kinetic folding data for Apaf-1 CARD and procaspase-3 provide an improved picture of the function and regulation of apoptosis. These studies propose new targets for therapeutic design to combat diseases associated with apoptosis. en_US
dc.rights I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dis sertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to NC State University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. en_US
dc.subject apoptosis en_US
dc.subject caspase en_US
dc.subject protein folding en_US
dc.subject kinetics en_US
dc.title Kinetic Folding Studies of Apaf-1 CARD and Procaspase-3 en_US
dc.degree.name PhD en_US
dc.degree.level dissertation en_US
dc.degree.discipline Biochemistry en_US


Files in this item

Files Size Format View
etd.pdf 4.430Mb PDF View/Open

This item appears in the following Collection(s)

Show simple item record