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Rapid folding and unfolding of Apaf-1 CARD

Sara L Milam1, Nathan I Nicely, Brett Feeney

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

Journal of Molecular Biology
|April 6, 2007
PubMed

Insights

The folding of Apaf-1 CARD, a key protein in apoptosis, reveals complex mechanisms beyond simple two-state models. Its secondary structure is less stable than its tertiary structure, with rapid folding involving parallel pathways.

Area of Science:

  • Biochemistry
  • Structural Biology
  • Molecular Biology

Background:

  • Caspase recruitment domains (CARDs) are crucial protein interaction modules.
  • CARDs belong to the death domain superfamily, characterized by a specific alpha-helical structure.
  • Apaf-1 CARD is a key component in the apoptotic protease activating factor 1.

Purpose of the Study:

  • To investigate the equilibrium and kinetic folding mechanisms of the Apaf-1 CARD.
  • To determine if a simple two-state model adequately describes Apaf-1 CARD folding.
  • To elucidate the role of unfolded conformations and intermediates in Apaf-1 CARD folding.

Main Methods:

  • Equilibrium unfolding studies at pH 6 and pH 8.
  • Kinetic folding and unfolding studies using single mixing and sequential mixing stopped-flow techniques.
  • Kinetic simulations to model the folding pathways.

Main Results:

  • A two-state equilibrium folding mechanism is insufficient for Apaf-1 CARD, indicating the presence of intermediates.
  • Secondary structure of Apaf-1 CARD is less stable than its tertiary structure.
  • Apaf-1 CARD folding and unfolding are rapid, involving parallel pathways with multiple unfolded conformations.

Conclusions:

  • Apaf-1 CARD folding is a complex process involving multiple unfolded states and intermediates.
  • The native ensemble of Apaf-1 CARD is formed rapidly during refolding.
  • This contrasts with other CARDs where folding is often hindered by kinetic traps.

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