Glutathione-dependent pathways of refolding of RNase T1 by oxidation and disulfide isomerization: catalysis by protein disulfide isomerase.
Protein folding, associated with oxidation and isomerization of disulfide bonds, was studied using reduced and denatured RNase T1 (rd-RNase T1) and mixed disulfide between glutathione and reduced RNase T1 (GS-RNase T1) as starting materials. Folding was initiated by addition of free glutathione (GSH + GSSG) and was monitored by electrospray mass spectrometry (ES-MS) time-course analysis. This permitted both the identification and quantitation of the population of intermediates present during the refolding process. Refolding experiments were performed in the presence of different absolute concentrations of glutathione species while keeping the redox potential fixed, in order to evaluate the effect of the glutathione concentration on the distribution of the refolding intermediates. All the analyses indicate a pathway of sequential reactions in the formation of native RNase T1 which occurs via the reiteration of two steps: (i) formation of a species containing both mixed disulfides with glutathione and free protein thiols, and (ii) formation of an intramolecular disulfide via thiol-disulfide interchange reaction between them. Refolding of rd-RNase T1 and GS-RNase T1 was also performed in the presence of protein disulfide isomerase (PDI). Addition of PDI led to a catalysis of each individual reaction of the entire process without altering the refolding pathway. Refolding reactions carried out at different absolute concentrations of glutathione proved that GSH and/or GSSG participate directly in the reaction catalyzed by PDI. On the basis of these experiments and previous results on the refolding of RNase A [Torella, C., Ruoppolo, M., Marino, G., & Pucci, P. (1994) FEBS Lett. 352, 301-306], a hypothesis of a general pathway for folding of S--S containing proteins is proposed.
Ruoppolo
M
M
Servizio Spettrometria di Massa, CNR-Università di Napoli, Naples, Italy.
Freedman
R B
RB
Pucci
P
P
Marino
G
G
D000818:Animals; D002417:Cattle; D004220:Disulfides / Q000378:metabolism*; D005978:Glutathione / Q000737:chemistry* / Q000378:metabolism; D007535:Isomerases / Q000378:metabolism*; D007536:Isomerism; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D013058:Mass Spectrometry; D008970:Molecular Weight; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011489:Protein Denaturation; D019704:Protein Disulfide-Isomerases; D017510:Protein Folding; D006163:Ribonuclease T1 / Q000737:chemistry* / Q000378:metabolism*
D004220:Disulfides; D006163:Ribonuclease T1; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D005978:Glutathione