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pH effects on the stability and dimerization of procaspase-3

Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, North Carolina 27695-7622, USA

(RECEIVED July 22, 2004; FINAL REVISION September 1, 2004; ACCEPTED September 3, 2004)

pH-dependent conformational changes are known to occur in dimeric procaspase-3, and they have been shown to affect the rate of automaturation. We studied the equilibrium unfolding of procaspase-3(C163S) as a function of pH (between pH 8.5 and pH 4) in order to examine these changes in the context of folding and stability. The data show that the procaspase dimer undergoes a pH-dependent dissociation below pH 5, so that the protein is mostly monomeric at pH 4. Consistent with this, the dimer unfolds via a four-state process between pH 8.5 and pH 4.75, in which the native dimer isomerizes to a dimeric intermediate, and the dimeric intermediate dissociates to a monomer, which then unfolds. In contrast, a small protein concentration dependence was observed by circular dichroism, but not by fluorescence emission, at pH 4.5 and pH 4.2. There was no protein-concentration dependence to the data collected at pH 4. Overall, the results are consistent with the redistribution of the population of native dimer (N₂) to dimeric intermediate (I₂) to monomeric intermediate (I), as the pH is lowered so that at pH 4, the "native" ensemble resembles the monomeric intermediate (I) observed during unfolding at higher pH. An emerging picture of the monomeric procaspase is discussed. Procaspase-3 is most stable at pH ~7 (24–26 kcal/mol), and while the stability decreased with pH, it was observed that dimerization contributes the majority (>70%) of the conformational free energy.

Keywords: caspase; dimerization; apoptosis; protein folding; fluorescence spectroscopy; circular dichroism

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041003305.

Reprint requests to: A. Clay Clark, Department of Molecular and Structural Biochemistry, 128 Polk Hall, North Carolina State University, Raleigh, NC 27695-7622, USA: e-mail: clay_clark{at}ncsu.edu; fax: (919) 515-2047.

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