Abstract
A comparative study of the oxidative refolding for nine selected potato carboxypeptidase inhibitor (PCI) mutants was carried out using the disulfide quenching approach. The mutations were performed at the N- and C-terminal tails of PCI outside its disulfide stabilized central core. The differences between the refolding of wild type and mutant proteins were observed in the second phase of the refolding process, the reshuffling of disulfide bridges, although the first phase, nonspecific packing, was not greatly affected by the mutations. Point mutations at the C-tail or deletion of up to three C-terminal residues of PCI resulted in a lower efficiency of the reshuffling process. In the case of the mutants lacking five N-terminal or four or five C-terminal residues, no "native-like" form was observed after the refolding process. On the other hand, the double mutant G35P/P36G did not attain a native-like form either, although one slightly more stable species was observed after being submitted to refolding. The disulfide pairing of this species is different from that of the wtPCI native form. The differences between the refolding process of wild type and mutant forms are interpreted in the light of the new view of protein folding. The results of the present study support the hypothesis that the refolding of this small disulfide-rich protein, and others, is driven by noncovalent interactions at the reshuffling stage. It is also shown that the interactions established between the N- and C-tail residues and the core of PCI are important for the proper refolding of the protein.
Highlights
Small proteins that are rich in disulfide bridges are good models for studying the folding process using the disulfide quenching method, where the folding intermediates that form during their oxidative refolding are trapped at low pH, separated on HPLC,1 and analyzed [1, 2]
As in the case of wtPCI, in the presence of cysteine, the equilibrated mixture of scrambled forms in such mutants decreased to give rise to the native-like species
The mutations that have the strongest influence on the folding of potato carboxypeptidase inhibitor (PCI) are the deletion of five residues at the N terminus or four or five residues at the C terminus, giving rise to a ”trimmed“ core with 2/5, 7/1, and 7/0 residues at the N/C tails, respectively
Summary
Small proteins that are rich in disulfide bridges are good models for studying the folding process using the disulfide quenching method, where the folding intermediates that form during their oxidative refolding are trapped at low pH, separated on HPLC,1 and analyzed [1, 2]. Refolding Behavior—The oxidative refolding in vitro of different N- and C-tail mutant forms of PCI was studied by reverse-phase HPLC analysis of the acid-trapped disulfide intermediates present in samples collected at different times during the refolding processes, these processes being followed either in the absence or presence of an external thiol added (cysteine) (see “Experimental Procedures”).
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.
