Premium
Mutations in domain a ′ of protein disulfide isomerase affect the folding pathway of bovine pancreatic ribonuclease A
Author(s) -
Ruoppolo Margherita,
Orrù Stefania,
Talamo Fabio,
Ljung Johanna,
Pirneskoski Annamari,
Kivirikko Kari I.,
Marino Gennaro,
Koivunen Peppi
Publication year - 2003
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.0242803
Subject(s) - protein disulfide isomerase , bovine pancreatic ribonuclease , rnase p , protein folding , chemistry , ribonuclease , microsomal triglyceride transfer protein , chaperone (clinical) , biochemistry , isomerase , mutant , protein subunit , folding (dsp implementation) , enzyme , rna , lipoprotein , medicine , pathology , very low density lipoprotein , cholesterol , gene , electrical engineering , engineering
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, Δ455–457, and abb ′, and the individual domains a and a ′. The first two mutants contained alterations in the last α 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 Δ455–457, 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.