title: Rapid folding and unfolding of Apaf-1 CARD.
abstract: Caspase recruitment domains (CARDs) are members of the death domain superfamily and contain six antiparallel helices in an alpha-helical Greek key topology. We have examined the equilibrium and kinetic folding of the CARD of Apaf-1 (apoptotic protease activating factor 1), which consists of 97 amino acid residues, at pH 6 and pH 8. The results showed that an apparent two state equilibrium mechanism is not adequate to describe the folding of Apaf-1 CARD at either pH, suggesting the presence of intermediates in equilibrium unfolding. Interestingly, the results showed that the secondary structure is less stable than the tertiary structure, based on the transition mid-points for unfolding. Single mixing and sequential mixing stopped-flow studies showed that Apaf-1 CARD folds and unfolds rapidly and suggest a folding mechanism that contains parallel channels with two unfolded conformations folding to the native conformation. Kinetic simulations show that a slow folding phase is described by a third conformation in the unfolded ensemble that interconverts with one or both unfolded species. Overall, the native ensemble is formed rapidly upon refolding. This is in contrast to other CARDs in which folding appears to be dominated by formation of kinetic traps.
authors.affiliation: Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695, USA.
authors.forename: Sara L
authors.initials: SL
authors.lastname: Milam
authors.forename: Nathan I
authors.initials: NI
authors.lastname: Nicely
authors.forename: Brett
authors.initials: B
authors.lastname: Feeney
authors.forename: Carla
authors.initials: C
authors.lastname: Mattos
authors.forename: A Clay
authors.initials: AC
authors.lastname: Clark
mesh_terms: D053477:Apoptotic Protease-Activating Factor 1 / Q000737:chemistry* / Q000378:metabolism*; D018360:Crystallography, X-Ray; D005453:Fluorescence; D007700:Kinetics; D008954:Models, Biological; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013816:Thermodynamics
chemical_list: D053477:Apoptotic Protease-Activating Factor 1