PROLYL ISOMERASE AS A PROBE OF STABILITY OF SLOW-FOLDING INTERMEDIATES

Catalysis of slow folding reactions by peptidyl prolyl cis-trans isome rase (PPI) provides estimates of stabilities of intermediates in foldi ng of normal and mutational variants of yeast iso-2 cytochrome c. A tw o-state model postulating a rapid preequilibration of intermediates wi th the unfolded protein is employed to calculate the stabilization fre e energy of the intermediate from the catalytic efficiency (k(cat)/K-m ) of PPI toward slow folding species. Stability measurements have been made for two distinct slow-folding intermediates: the absorbance-dete cted (I(I)s) and fluorescence-detected (I(II)s) intermediates. Mutatio n-induced changes in the stability of the intermediates and in the act ivation free energy for slow folding are compared to changes in equili brium thermodynamic stability. The results show that (1) for iso-2 the absorbance-detected intermediates (I(I)s) are slightly more stable th an the fluorescence-detected intermediates (I(II)s), (2) most mutation s have different effects on equilibrium stability and the stability of the I(I)s or I(II)s intermediates, and (3) for both slow folding reac tions the mutation-induced changes in the activation free energy are s mall compared to the magnitude of the activation free energy barrier. Differential effects of mutations on equilibrium stability and the sta bility of intermediates provides a means of assessing the sequence-enc oded structural specificity for folding. Mutations with different effe cts on intermediate stability and equilibrium stability change the enc oded folding information and may alter folding pathways and/or lead to different three-dimensional structures. Identification of mutations w hich stabilize a folding intermediate relative to the native conformat ion provides an empirical approach to the design of thermodynamically stable forms of folding intermediates.