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
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引用次数: 11

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.

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马铃薯羧肽酶抑制剂前序列对成熟蛋白折叠的影响分析。
蛋白质折叠在体内可受多种因素调节。虽然伴侣蛋白作为折叠催化剂,显示出广泛的底物特异性,但一些前肽特异性地促进了它们所结合的成熟蛋白的折叠。马铃薯羧肽酶抑制剂(Potato carboxypeptidase inhibitor, PCI)是一种含有27个残基n端和7个残基c端前区的前体蛋白,是一种具有39个残基的羧肽酶抑制剂。在这项工作中,成熟的PCI在体外的二硫偶联折叠已经与用N端前序列(ProNtPCI)或N端和c端前序列(ProPCI)延伸的相同蛋白质的折叠进行了比较,也与反式中的N端前序列(ProNt + PCI)进行了比较。在所有这些分子的折叠动力学或效率方面没有观察到显着差异。此外,在大肠杆菌中使用野生型PCI和三种具有或不具有n端前序列的PCI突变形式进行了体内折叠研究,这些突变先前已报道影响成熟形式的PCI折叠。每个结构向介质分泌的“原生样”形式的程度不受n端前序列存在的影响。这些结果表明,在体外和体内大肠杆菌中,PCI的折叠不依赖于n端前序列。然而,通过光谱、氢交换和质谱有限蛋白水解的结构分析表明,这种n端前序列能够在前体形式内折叠。
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