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Kinetic traps in the folding/unfolding of procaspase-1 CARD domain

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

(RECEIVED November 18, 2003; FINAL REVISION April 23, 2004; ACCEPTED May 16, 2004)

We have examined the folding and unfolding of the caspase recruitment domain of procaspase-1 (CP1-CARD), a member of the -helical Greek key protein family. The equilibrium folding/unfolding of CP1-CARD is described by a two-state mechanism, and the results show CP1-CARD is marginally stable with a G^H₂O of 1.1 ± 0.2 kcal/mole and an m-value of 0.65 ± 0.06 kcal/mole/M (10 mM Tris-HCl at pH 8.0, 1 mM DTT, 25°C). Consistent with the equilibrium folding data, CP1-CARD is a monomer in solution when examined by size exclusion chromatography. Single-mixing stopped-flow refolding and unfolding studies show that CP1-CARD folds and unfolds rapidly, with no detectable slow phases, and the reactions appear to reach equilibrium within 10 msec. However, double jump kinetic experiments demonstrate the presence of an unfolded-like intermediate during unfolding. The intermediate converts to the fully unfolded conformation with a half-time of 10 sec. Interrupted refolding studies demonstrate the presence of one or more nativelike intermediates during refolding, which convert to the native conformation with a half-time of about 60 sec. Overall, the data show that both unfolding and refolding processes are slow, and the pathways contain kinetically trapped species.

Keywords: caspase recruitment domain; -helical Greek key; kinetic trap; double jump; interrupted re-folding; equilibrium folding

Abbreviations: CARD, caspase recruitment domain • CP1-CARD, CARD of procaspase-1 • RICK, RIP-like interacting CLARP kinase • DD, death domain • DED, death effector domain • APAF-1, apoptotic protease activating factor • IPTG, isopropyl -D-1-thiogalactopyranoside • TCEP, Tris(2-carboxyethyl)-phosphine hydrochloride • CO, relative contact order • LRO, long range order

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

¹ Present address: Department of Molecular Biology and Biochemistry, University of California at Irvine, Irvine, CA 92697, USA.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03521504.

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				Y.-R. Chen and A. C. Clark Substitutions of prolines examine their role in kinetic trap formation of the caspase recruitment domain (CARD) of RICK. Protein Sci., March 1, 2006; 15(3): 395 - 409. [Abstract] [Full Text] [PDF]