Folding, Stability and Active Site Conformation of Procaspase-3

procaspase-3;folding active site

Bose, Kakoli ; Clay Clark, Committee Chair,Bose, Kakoli ; Clay Clark ; Committee Chair

University:North Carolina State University

关键词: procaspase-3;    folding active site;   

Others  :  https://repository.lib.ncsu.edu/bitstream/handle/1840.16/5688/etd.pdf?sequence=1&isAllowed=y

美国|英语

来源: null

【 摘 要 】

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 Km 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).