Protein disulfide isomerase exhibits chaperone and anti-chaperone activity in the oxidative refolding of lysozyme.
Reduced, denatured lysozyme tends to aggregate at neutral pH, and competition between productive folding and aggregation substantially reduces the efficiency of refolding (Goldberg, M.E., Rudolph, R., and Jaenicke, R. (1991) Biochemistry 30, 2790-2797). Protein disulfide isomerase (PDI), a catalyst of oxidative protein folding, has a variety of effects on the yield of native lysozyme during the oxidative refolding of the reduced, denatured protein. Depending on the concentration of lysozyme, the concentration of PDI, and the order in which lysozyme and PDI are added to initiate folding, PDI can produce a substantial increase or a substantial decrease in the recovery of native lysozyme, when compared with the uncatalyzed reaction. In the presence of a glutathione redox buffer, denatured lysozyme (1-10 microM) partitions almost equally between productive folding leading to native lysozyme (50-63%) and non-productive fates including the formation of disulfide cross-linked aggregates. At the higher lysozyme concentrations examined (5-10 microM), substoichiometric concentrations of PDI (0.5-1 microM) exhibit "anti-chaperone" activity; PDI actively diverts most of the denatured lysozyme away from productive folding so that only 17 +/- 9% of the lysozyme is recovered as native enzyme. PDI's anti-chaperone activity results in extensive intermolecular disulfide crosslinking of lysozyme into large, inactive aggregates. On the other hand, if PDI is initially present at a large molar excess (5-10-fold) when denatured lysozyme is diluted to initiate folding, PDI demonstrates a chaperone-like activity that prevents aggregate formation and promotes correct folding. When PDI's chaperone activity is dominant, virtually all of the denatured lysozyme is correctly folded. The schizophrenic chaperone/anti-chaperone nature of PDI activity accounts for a number of observations on in vivo protein folding, including the necessity for maintaining a high concentration of PDI in the endoplasmic reticulum and the formation of disulfide cross-linked aggregates in the endoplasmic reticulum during the expression of disulfide-containing proteins (deSilva, A., Braakman, I., and Helenius, A. (1993) J. Cell. Biol. 120, 647-655).
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Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030.
Gilbert
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HF
D000818:Animals; D002384:Catalysis; D018833:Chaperonins; D002645:Chickens; D007535:Isomerases / Q000378:metabolism*; D007700:Kinetics; D009113:Muramidase / Q000737:chemistry / Q000378:metabolism*; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D019704:Protein Disulfide-Isomerases; D017510:Protein Folding; D011506:Proteins / Q000378:metabolism*
D011506:Proteins; D009113:Muramidase; D018833:Chaperonins; D007535:Isomerases; D019704:Protein Disulfide-Isomerases