title: Mutations in domain a' of protein disulfide isomerase affect the folding pathway of bovine pancreatic ribonuclease A.
abstract: Protein disulfide isomerase (PDI, EC 5.3.4.1), an enzyme and chaperone, catalyses disulfide bond formation and rearrangements in protein folding. It is also a subunit in two proteins, the enzyme collagen prolyl 4-hydroxylase and the microsomal triglyceride transfer protein. It consists of two catalytically active domains, a and a', and two inactive ones, b and b', all four domains having the thioredoxin fold. Domain b' contains the primary peptide binding site, but a' is also critical for several of the major PDI functions. Mass spectrometry was used here to follow the folding pathway of bovine pancreatic ribonuclease A (RNase A) in the presence of three PDI mutants, F449R, Delta455-457, and abb', and the individual domains a and a'. The first two mutants contained alterations in the last alpha helix of domain a', while the third lacked the entire domain a'. All mutants produced genuine, correctly folded RNase A, but the appearance rate of 50% of the product, as compared to wild-type PDI, was reduced 2.5-fold in the case of PDI Delta455-457, 7.5-fold to eightfold in the cases of PDI F449R and PDI abb', and over 15-fold in the cases of the individual domains a and a'. In addition, PDI F449R and PDI abb' affected the distribution of folding intermediates. Domains a and a' catalyzed the early steps in the folding but no disulfide rearrangements, and therefore the rate observed in the presence of these individual domains was similar to that of the spontaneous process.
authors.affiliation: Dipartimento di Biochimica e Biotecnologie Mediche, School of Biotechnological Sciences, Università degli Studi di Napoli Federico II, Napoli, Italy. ruoppolo@dbbm.unina.it
authors.forename: Margherita
authors.initials: M
authors.lastname: Ruoppolo
authors.forename: Stefania
authors.initials: S
authors.lastname: Orrù
authors.forename: Fabio
authors.initials: F
authors.lastname: Talamo
authors.forename: Johanna
authors.initials: J
authors.lastname: Ljung
authors.forename: Annamari
authors.initials: A
authors.lastname: Pirneskoski
authors.forename: Kari I
authors.initials: KI
authors.lastname: Kivirikko
authors.forename: Gennaro
authors.initials: G
authors.lastname: Marino
authors.forename: Peppi
authors.initials: P
authors.lastname: Koivunen
mesh_terms: D000818:Animals; D002417:Cattle; D007700:Kinetics; D018832:Molecular Chaperones / Q000737:chemistry / Q000235:genetics; D009154:Mutation; D019704:Protein Disulfide-Isomerases / Q000737:chemistry* / Q000235:genetics; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012259:Ribonuclease, Pancreatic / Q000737:chemistry*
chemical_list: D018832:Molecular Chaperones; D012259:Ribonuclease, Pancreatic; D019704:Protein Disulfide-Isomerases