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

Yun-Ru Chen1, A Clay Clark

  • 1Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, North Carolina 27695, USA.

Protein Science : a Publication of the Protein Society
|July 27, 2004
PubMed

Insights

The caspase recruitment domain of procaspase-1 (CP1-CARD) is marginally stable and folds/unfolds rapidly. However, kinetic studies reveal slow unfolding and refolding pathways with trapped intermediates.

Area of Science:

  • Protein folding and dynamics
  • Biochemistry
  • Molecular biology

Background:

  • The caspase recruitment domain of procaspase-1 (CP1-CARD) is a key component of the inflammasome.
  • Understanding CP1-CARD's folding is crucial for its function and regulation.
  • CP1-CARD belongs to the alpha-helical Greek key protein family.

Purpose of the Study:

  • To investigate the folding and unfolding mechanisms of CP1-CARD.
  • To characterize the thermodynamic stability and kinetic properties of CP1-CARD.
  • To identify any intermediates present during the folding/unfolding pathways.

Main Methods:

  • Equilibrium denaturation studies using guanidine hydrochloride (implied by DeltaG and m-value).
  • Size exclusion chromatography to determine oligomeric state.
  • Stopped-flow spectroscopy (single-mixing for refolding/unfolding, double-jump for kinetics, interrupted refolding).

Main Results:

  • CP1-CARD exhibits marginal stability (DeltaG(H2O) = 1.1 +/- 0.2 kcal/mole) and is a monomer in solution.
  • Fast folding/unfolding kinetics (equilibrium within 10 msec) were observed initially.
  • Kinetic experiments revealed slow unfolding (10 sec halftime for intermediate to unfolded) and refolding (60 sec halftime for intermediates to native).

Conclusions:

  • CP1-CARD folding/unfolding pathways are complex and deviate from a simple two-state model.
  • Kinetically trapped intermediates exist during both unfolding and refolding processes.
  • These findings highlight the dynamic nature of CP1-CARD and potential regulatory mechanisms.

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