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Folding, stability and active site c...

Bose, Kakoli.

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Folding, stability and active site conformation of procaspase-3.

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	Folding, stability and active site conformation of procaspase-3./
作者:	Bose, Kakoli.
面頁冊數:	204 p.
附註:	Source: Dissertation Abstracts International, Volume: 64-11, Section: B, page: 5397.
Contained By:	Dissertation Abstracts International64-11B.
標題:	Biophysics, General. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3112793

Folding, stability and active site conformation of procaspase-3.
Bose, Kakoli.

Folding, stability and active site conformation of procaspase-3. - 204 p.

Source: Dissertation Abstracts International, Volume: 64-11, Section: B, page: 5397.

Thesis (Ph.D.)--North Carolina State University, 2004.

We have examined the folding, assembly and active site conformation of a catalytically inactive mutant of procaspase-3 (procaspase-3(C163S)), a homodimeric protein that belongs to the caspase family of proteases. The caspase family, and especially caspase-3, is integral to apoptosis. We show that an uncleavable mutant (D3A) of procaspase-3 is catalytically active with K m similar to that of its mature counterpart. We developed limited proteolysis assays using trypsin and V8 proteases, which allow the examination of amino acids in three of five active site loops. In addition, we examined the response of the two tryptophanyl residues in the active site to several quenching agents over the pH range of 3 to 9. Overall, the data suggest that the major conformational change that occurs upon maturation results in formation of the loop bundle between loops L4, L2 and L2'. The pKa's of both catalytic groups decrease as a result of the loop movements. However, loop L3, which comprises the bulk of the substrate-binding pocket, does not appear to be unraveled and solvent exposed, even at lower pH. In order to understand the active site formation in the context of folding, we looked into the equilibrium unfolding of procaspase-3(C163S). The equilibrium unfolding of procaspase-3(C163S) is described by a four-state equilibrium model in which the native dimer undergoes an isomeration to a dimeric intermediate, and the dimeric intermediate dissociates to a monomeric intermediate, which then unfolds. Therefore, dimerization is a folding event and it contributes significantly to the protein stability (18.8 kcal/mol of 25.8 kcal/mol). Equilibrium unfolding experiments of procaspase-3 (C163S) at different pH shows maximum stability at pH 7.2, and a transition from four-state model to a three-state monomer and finally to a two-state monomer model with decrease in pH. This is representative of conformational change and dimer dissociation at lower pH (between pH 5 and 4). The pro-less variant of procaspase-3(C163S) folds reversibly only in 1:1 protein to pro-peptide ratio suggesting it might act as an intramolecular chaperone (IMC).Subjects--Topical Terms:

1019105
Biophysics, General.

Folding, stability and active site conformation of procaspase-3.
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520 $a We have examined the folding, assembly and active site conformation of a catalytically inactive mutant of procaspase-3 (procaspase-3(C163S)), a homodimeric protein that belongs to the caspase family of proteases. The caspase family, and especially caspase-3, is integral to apoptosis. We show that an uncleavable mutant (D3A) of procaspase-3 is catalytically active with K m similar to that of its mature counterpart. We developed limited proteolysis assays using trypsin and V8 proteases, which allow the examination of amino acids in three of five active site loops. In addition, we examined the response of the two tryptophanyl residues in the active site to several quenching agents over the pH range of 3 to 9. Overall, the data suggest that the major conformational change that occurs upon maturation results in formation of the loop bundle between loops L4, L2 and L2'. The pKa's of both catalytic groups decrease as a result of the loop movements. However, loop L3, which comprises the bulk of the substrate-binding pocket, does not appear to be unraveled and solvent exposed, even at lower pH. In order to understand the active site formation in the context of folding, we looked into the equilibrium unfolding of procaspase-3(C163S). The equilibrium unfolding of procaspase-3(C163S) is described by a four-state equilibrium model in which the native dimer undergoes an isomeration to a dimeric intermediate, and the dimeric intermediate dissociates to a monomeric intermediate, which then unfolds. Therefore, dimerization is a folding event and it contributes significantly to the protein stability (18.8 kcal/mol of 25.8 kcal/mol). Equilibrium unfolding experiments of procaspase-3 (C163S) at different pH shows maximum stability at pH 7.2, and a transition from four-state model to a three-state monomer and finally to a two-state monomer model with decrease in pH. This is representative of conformational change and dimer dissociation at lower pH (between pH 5 and 4). The pro-less variant of procaspase-3(C163S) folds reversibly only in 1:1 protein to pro-peptide ratio suggesting it might act as an intramolecular chaperone (IMC).
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