Equilibrium and Kinetic folding properties of alpha-helical Greek key protein domains

Abstract

I have characterized the equilibrium and kinetic folding properties of members of alpha-helical Greek key protein domain, caspase recruitment domains of RICK (RICK-CARD) and procaspase-1 (Pro-1-CARD).

At equilibrium, folding of both RICK-CARD and Pro-1-CARD is well described by a two-state mechanism representing the native and unfolded ensembles. The proteins are marginally stable,with the Gibbs free energy of 3.0 and 1.1 kcal/mol and m-values of 1.27 and 0.68 kcal/mol/M for RICK-CARD and Pro-1-CARD, respectively (30 mM Tris-HCl, pH 8, 1 mM DTT, 25 degree Celsius).

The folding pathways of RICK-CARD are complex and contain at least two or three non-native conformations. The major folding event of RICK-CARD has a folding rate constant of 30 s⁻¹.

Studies on seven mutants of RICK-CARD suggest that the three prolyl residues contribute to its structural stability and folding complexity. The folding of Pro-1-CARD is different than that of RICK-CARD. The folding and unfolding kinetics of Pro-1-CARD are fast but with kinetically trapped intermediates present, which appear to be similar to that of RICK-CARD. The work suggests that different alpha-helical Greek key protein domains fold differently but may share some similar folding pathways.