Mutations in the N- and C-terminal tails of potato carboxypeptidase inhibitor influence its oxidative refolding process at the reshuffling stage.
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.
Venhudova
G
G
Institut de Biologia Fonamental and Departament de Bioquimica i Biologia Molecular, Universitat Autonoma de Barcelona, 08193 Bellaterra (Barcelona), Spain.
Canals
F
F
Querol
E
E
Aviles
F X
FX
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D009154:Mutation; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011480:Protease Inhibitors; D017510:Protein Folding; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D010940:Plant Proteins; D011480:Protease Inhibitors