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.