Abstract
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.
Highlights
The rank order of chain folding, prolyl isomerization, and disulfide bond formation can vary in the oxidative folding of ribonuclease Tl
We ask whether the formation of ordered structure, the introduction of disulfide bonds, and the isomerization of prolyl peptide bonds have to occur in a defined order to warrant productive folding and an efficient function of the folding enzymes
We find that the oxidative folding of C2S/C10N-RNase T1 can be extremely rapid when both DsbA and prolyl isomerase are present
Summary
The rank order of chain folding, prolyl isomerization, and disulfide bond formation can vary in the oxidative folding of ribonuclease Tl. Our in vitro experiments are carried out by using the thiol-disulfide oxidoreductase DsbA (Bardwell et al, 1991; Kamitani et al, 1992; Akiyama et al, 1992; Bardwell and Beckwith, 1993; Zapun and Creighton, 1994; Nelson and Creighton, 1994) as the oxidant and prolyl isomerase A (Hayano et al, 1991) as the catalyst ofprolyl isomerization. Both are from the periplasm of Escherichia coli
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
