Influence of protein conformation on disulfide bond formation in the oxidative folding of ribonuclease T1.
In oxidative protein folding the interdependence between the acquisition of an ordered native-like conformation and disulfide bond formation was investigated by using the C2S/C10N variant of ribonuclease T1 as a model. This protein of 104 residues has a single disulfide bond between Cys6 and Cys103. In the reduced form it is unfolded in the presence of urea, but native-like folded when > or = 1.5 M NaCl is present. The influence of a folded conformation on the individual thiol/disulfide exchange reactions between the protein and glutathione could thus be studied in oxidative folding by varying the urea and NaCl concentrations. When the reduced protein was folded native-like the initial formation of the mixed disulfide between the protein and glutathione was decelerated about fourfold. The attachment of a glutathionyl moiety in this step destabilizes the protein by about 5 kJ mol-1 and led to a local unfolding near the two Cys residues. The reacting thiol groups still remained in close proximity for the subsequent intramolecular thiol/disulfide exchange reaction, but an increase in the energy of the transition state (e.g. by a hydrophobic environment or by steric strain) could be avoided. As a consequence the formation of the protein disulfide in this reaction was 100-fold faster when the mixed-disulfide species was in this ordered conformation. These results illustrate the importance of a low stability and a high flexibility of folding intermediates.
Frech
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Laboratorium für Biochemie, Universität Bayreuth, Germany.
Schmid
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D001236:Aspergillus oryzae / Q000201:enzymology; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D010084:Oxidation-Reduction; D011487:Protein Conformation; D017510:Protein Folding; D006163:Ribonuclease T1 / Q000737:chemistry* / Q000378:metabolism*; D012997:Solvents; D013997:Time Factors
D004220:Disulfides; D012997:Solvents; D006163:Ribonuclease T1; D005978:Glutathione; D003545:Cysteine