CHARACTERIZATION OF FOLDING INTERMEDIATES USING PROLYL ISOMERASE

Structure-reactivity relationships of human peptidyl prolyl cis-trans isomerase (PPI) toward the two slow folding reactions of yeast iso-2 c ytochrome c have been used to characterize the structure of folding in termediates in the vicinity of critical prolines. We propose that the relative catalytic efficiency of PPI for the protein substrate relativ e to a peptide substrate, (k(cat)/K(m))rel, is a measure of structure in folding intermediates. The structural stability of slow-folding int ermediates as detected by changes in (k(cat)/K(m))rel was investigated using two structural perturbants: guanidine hydrochloride and site-di rected mutagenesis. Neither of the two slow folding reactions for wild -type cytochrome c is catalyzed at low denaturant concentrations. Howe ver, both phases are catalyzed at moderate concentrations of guanidine hydrochloride. A mutation in cytochrome c enhances catalysis of the f luorescence-detected slow folding phase. For protein substrates destab ilized by denaturants or mutation, we suggest that increases in (k(cat )/K(m))rel result from a loosening of the substrate structure, providi ng better access of peptidyl prolyl isomerase to critical proline(s).