NC State Theses and Dissertations
Permanent URI for this communityhttps://www.lib.ncsu.edu/resolver/1840.20/23
Browse
Browsing NC State Theses and Dissertations by Advisor "A. Clay Clark, Committee Chair"
Now showing 1 - 3 of 3
- Results Per Page
- Sort Options
- Equilibrium and Kinetic folding properties of alpha-helical Greek key protein domains(2003-11-13) Chen, Yun-Ru; Carla Mattos, Committee Member; A. Clay Clark, Committee Chair; Harold Swaisgood, Committee Member; Dennis T. Brown, Committee MemberI have characterized the equilibrium and kinetic folding properties of members of alpha-helical Greek key protein domain, caspase recruitment domains of RICK (RICK-CARD) and procaspase-1 (Pro-1-CARD). At equilibrium, folding of both RICK-CARD and Pro-1-CARD is well described by a two-state mechanism representing the native and unfolded ensembles. The proteins are marginally stable,with the Gibbs free energy of 3.0 and 1.1 kcal/mol and m-values of 1.27 and 0.68 kcal/mol/M for RICK-CARD and Pro-1-CARD, respectively (30 mM Tris-HCl, pH 8, 1 mM DTT, 25 degree Celsius). The folding pathways of RICK-CARD are complex and contain at least two or three non-native conformations. The major folding event of RICK-CARD has a folding rate constant of 30 s⁻¹. Studies on seven mutants of RICK-CARD suggest that the three prolyl residues contribute to its structural stability and folding complexity. The folding of Pro-1-CARD is different than that of RICK-CARD. The folding and unfolding kinetics of Pro-1-CARD are fast but with kinetically trapped intermediates present, which appear to be similar to that of RICK-CARD. The work suggests that different alpha-helical Greek key protein domains fold differently but may share some similar folding pathways.
- Kinetic Folding Studies of Apaf-1 CARD and Procaspase-3(2008-11-13) Milam, Sara Lis; Carol Hall, Committee Member; William Miller, Committee Member; A. Clay Clark, Committee Chair; Michael Goshe, Committee MemberApoptosis 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.
- Structure, Folding, and Assembly of (Pro)caspase-3. The Role of the Dimer Interface in Active Site Formation.(2009-05-07) Walters, Jad Anthony; A. Clay Clark, Committee Chair; Carl Hall, Committee Member; Paul Wollenzien, Committee Member; Bob Rose, Committee MemberEffector procaspase-3 plays a vital role in carrying out the final steps of programmed cell death, leading to the destruction of the cell. Because dimerization of (pro)caspase-3 is essential for enzyme stability and activity, it is important to understand the structural details of the interactions at the dimer interface. We show a single site in the dimer interface of (pro)caspase-3 can be used to activate or inhibit the enzyme. The results presented here demonstrate activation of procaspase-3, in the absence of intersubunit linker cleavage, which generates a constitutively active, uninhibitable enzyme that is very efficient in killing both healthy and diseased mammalian cells. Moreover, inhibition may also be achieved utilizing the same site and the structural studies reveal two novel pathways of inhibition. Overall, these studies show how the interactions at the dimer interface in procaspase-3 are essential in formation of a competent active site. In addition to the procaspase-3 interface studies, several crystallographic studies are presented which are aimed at elucidating a structure of procaspase-3. Finally, a comprehensive protocol for carrying out equilibrium folding studies and determining conformational stabilities of macromolecules is provided. All together, this work has lead to exciting and novel discoveries in the field of apoptosis, including new mechanisms to selectively manipulate cell death.
