Analysis of the effect of potato carboxypeptidase inhibitor pro-sequence on the folding of the mature protein

Sílvia Bronsoms, Josep Villanueva, Francesc Canals, Enrique Querol, Francesc X. Aviles

Research output: Contribution to journalArticleResearchpeer-review

9 Citations (Scopus)

Abstract

Protein folding can be modulated in vivo by many factors. While chaperones act as folding catalysts and show broad substrate specificity, some pro-peptides specifically facilitate the folding of the mature protein to which they are bound. Potato carboxypeptidase inhibitor (PCI), a 39-residue protein carboxypeptidase inhibitor, is synthesized in vivo as a precursor protein that includes a 27-residue N-terminal and a seven-residue C-terminal pro-regions. In this work the disulfide-coupled folding of mature PCI in vitro has been compared with that of the same protein extended with either the N-terminal pro-sequence (ProNtPCI) or both N- and C-terminal pro-sequences (ProPCI), and also with the N-terminal pro-sequence in trans (ProNt + PCI). No significant differences can be observed in the folding kinetics or efficiencies of all these molecules. In addition, in vivo folding studies in Escherichia coli have been performed using wild-type PCI and three PCI mutant forms with and without the N-terminal pro-sequence, the mutations had been previously reported to affect folding of the PCI mature form. The extent to which the 'native-like' form was secreted to the media by each construction was not affected by the presence of the N-terminal pro-sequence. These results indicate that PCI does not depend on the N-terminal pro-sequence for its folding in both, in vitro and in vivo in E. coli. However, structural analysis by spectroscopy, hydrogen exchange and limited proteolysis by mass spectrometry, indicate the capability of such N-terminal pro-sequence to fold within the precursor form.
Original languageEnglish
Pages (from-to)3641-3650
JournalEuropean Journal of Biochemistry
Volume270
Issue number17
DOIs
Publication statusPublished - 1 Sep 2003

Keywords

  • Disulfide
  • Pro-region
  • Protease inhibitor
  • Protein folding
  • Structure

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