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

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Title: Kinetic Folding Studies of Apaf-1 CARD and Procaspase-3
Author: Milam, Sara Lis
Advisors: Carol Hall, Committee Member
William Miller, Committee Member
A. Clay Clark, Committee Chair
Michael Goshe, Committee Member
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.
Date: 2008-11-13
Degree: PhD
Discipline: Biochemistry

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