DsbA-mediated disulfide bond formation and catalyzed prolyl isomerization in oxidative protein folding.

The interrelationship between the acquisition of ordered structure, prolyl isomerization, and the formation of the disulfide bonds in assisted protein folding was investigated by using a variant of ribonuclease T1 (C2S/C10N-RNase T1) with a single disulfide bond and two cis-prolyl bonds as a model protein. The thiol-disulfide oxidoreductase DsbA served as the oxidant for forming the disulfide bond and prolyl isomerase A as the catalyst of prolyl isomerization. Both enzymes are from the periplasm of Escherichia coli. Reduced C2S/C10N-RNase T1 is unfolded in 0 M NaCl, but native-like folded in > or = 2 M NaCl. Oxidation of 5 microM C2S/C10N-RNase T1 by 8 microM DsbA (at pH 7.0, 25 degrees C) is very rapid with a t1/2 of about 10 s (the second-order rate constant is 7 x 10(3) s-1 M-1), irrespective of whether the reduced molecules are unfolded or folded. When they are folded, the product of oxidation is the native protein. When they are denatured, first the disulfide bond is formed in the unfolded protein chains and then the native structure is acquired. This slow reaction is limited in rate by prolyl isomerization and catalyzed by prolyl isomerase. The efficiency of this catalysis is strongly decreased by the presence of the disulfide bond. Apparently, the rank order of chain folding, prolyl isomerization, and disulfide bond formation can vary in the oxidative folding of ribonuclease T1. Such a degeneracy could generally be an advantage for protein folding both in vitro and in vivo.