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 cytochrome c have been used to characterize the structure of folding intermediates in the vicinity of critical prolines. We propose that the relative catalytic efficiency of PPI for the protein substrate relative to a peptide substrate, (kcat/Km)rel, is a measure of structure in folding intermediates. The structural stability of slow-folding intermediates as detected by changes in (kcat/Km)rel was investigated using two structural perturbants: guanidine hydrochloride and site-directed mutagenesis. Neither of the two slow folding reactions for wild-type cytochrome c is catalyzed at low denaturant concentrations. However, both phases are catalyzed at moderate concentrations of guanidine hydrochloride. A mutation in cytochrome c enhances catalysis of the fluorescence-detected slow folding phase. For protein substrates destabilized by denaturants or mutation, we suggest that increases in (kcat/Km)rel result from a loosening of the substrate structure, providing better access of peptidyl prolyl isomerase to critical proline(s).
Veeraraghavan
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Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
Nall
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D000591:Amino Acid Isomerases / Q000494:pharmacology*; D002352:Carrier Proteins / Q000494:pharmacology*; D002384:Catalysis; D003574:Cytochrome c Group / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D045304:Cytochromes c; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D008956:Models, Chemical; D009154:Mutation; D019696:Peptidylprolyl Isomerase; D011392:Proline / Q000378:metabolism*; D011489:Protein Denaturation; D017510:Protein Folding; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D013997:Time Factors
D002352:Carrier Proteins; D003574:Cytochrome c Group; D006146:Guanidines; C032139:iso-2-cytochrome C; D045304:Cytochromes c; D011392:Proline; D000591:Amino Acid Isomerases; D019696:Peptidylprolyl Isomerase; D019791:Guanidine