tailieunhanh - Báo cáo khoa học: Analysis of the effect of potato carboxypeptidase inhibitor pro-sequence on the folding of the mature protein

Protein folding can be modulatedin 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 pro-tein 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. | Eur. J. Biochem. 270 3641-3650 2003 FEBS 2003 doi Analysis of the effect of potato carboxypeptidase inhibitor pro-sequence on the folding of the mature protein Silvia Bronsoms Josep Villanueva Francesc Canals Enrique Querol and Francesc X. Aviles Institut de Biotecnologia i Biomedicina and Departament de Bioquimica i Biologia Molecular Universitat Autonoma de Barcelona Spain 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. .