Refolding by disulfide isomerization: the mixed disulfide between ribonuclease T1 and glutathione as a model refolding substrate.
Protein folding, associated with isomerization of disulfide bonds, was studied using the mixed disulfide between glutathione and reduced ribonuclease T1 (GS-RNase T1) as a stable soluble and homogeneous starting material; conditions were selected to model those within the lumen of the endoplasmic reticulum where native disulfide bonds are formed in protein biosynthesis. Folding was initiated by addition of free glutathione (GSH +/- GSSG) to promote thiol-disulfide interchange and was monitored by intrinsic protein fluorescence, appearance of native ribonuclease activity, HPLC, and nonreducing SDS-PAGE. All the analyses indicated that native RNase T1 was recovered in high yield in a variety of redox conditions. Appearance of native activity followed first-order kinetics; kinetic analysis of the intrinsic fluorescence changes indicated an additional rapid process in some conditions, interpreted as the formation of a nonnative intermediate state. Analysis by HPLC and SDS-PAGE also indicated the formation of transient intermediates. In 1.5 M NaCl, GS-RNase T1 adopts a compact native-like conformation; refolding by thiol-disulfide interchange in these conditions was accelerated approximately 2-fold. Refolding of GS-RNase T1 was catalyzed by protein disulfide isomerase (PDI); substoichiometric quantities of PDI accelerated refolding several-fold. GS-RNase T1 refolding was inhibited by BiP; refolding was completely blocked in presence of a 5-fold molar excess of BiP, and the yield of refolding was substantially reduced by equimolar concentrations of BiP; the refolding was then restored by the addition of ATP. GS-RNase T1 is a convenient model substrate for studying protein folding linked to native disulfide formation in conditions comparable to those within the lumen of the endoplasmic reticulum.
Ruoppolo
M
M
Research School of Biosciences, Biological Laboratory, University of Kent, Canterbury, U.K.
Freedman
R B
RB
D000818:Animals; D002417:Cattle; D004220:Disulfides; D004721:Endoplasmic Reticulum / Q000201:enzymology; D005978:Glutathione; D007535:Isomerases / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008958:Models, Molecular; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D019704:Protein Disulfide-Isomerases; D017510:Protein Folding; D017433:Protein Structure, Secondary; D006163:Ribonuclease T1 / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D004220:Disulfides; D006163:Ribonuclease T1; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D005978:Glutathione