Unfolding intermediates of the extracellular domain of the interferon gamma receptor.
Reduction of proteins which require disulfide bonds to be stable in the folded state is accompanied by step-wise unfolding. A soluble human interferon gamma receptor produced in Escherichia coli was used to investigate the kinetics of formation of unfolding intermediates. The protein includes 8 cysteine residues forming four disulfide bonds. It was reduced by using either dithiothreitol or the thioredoxin reduction system. Reduction with dithiothreitol resulted in formation of mainly four monomeric unfolding species as visualized by sodium dodecyl sulfate-polyacrylamide gels. The enzymatically catalyzed reaction produced only small amounts of two monomeric products and mostly delivered oligomeric and polymeric forms. In both cases, the ligand binding capacity of the receptor was significantly reduced immediately after appearance of the first intermediate. The intermediates involved interchange of disulfide bonds and did not show ligand binding capacity. Some of them were recognized by specific antibodies which detect conformational epitopes on the native interferon gamma receptor. On the basis of the antibody binding, a preliminary characterization of the formed intermediates was attempted. When the soluble receptor was reduced in the presence of denaturing agents, the reduction products were different from the unfolding intermediates generated in the absence of denaturants.
F. Hoffmann-La Roche Ltd., Pharmaceutical Research-New Technologies, Basel, Switzerland.
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Fountoulakis
D015153:Blotting, Western; D002384:Catalysis; D004220:Disulfides / Q000378:metabolism; D004229:Dithiothreitol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D019791:Guanidine; D006146:Guanidines / Q000378:metabolism; D006801:Humans; D007372:Interferons / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011971:Receptors, Immunologic / Q000378:metabolism*; D017471:Receptors, Interferon; D013880:Thioredoxin-Disulfide Reductase / Q000378:metabolism; D013879:Thioredoxins / Q000378:metabolism; D014508:Urea / Q000378:metabolism
D004220:Disulfides; D006146:Guanidines; D011971:Receptors, Immunologic; D017471:Receptors, Interferon; D013879:Thioredoxins; D009249:NADP; D014508:Urea; D007372:Interferons; D013880:Thioredoxin-Disulfide Reductase; D019791:Guanidine; D004229:Dithiothreitol