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Development of new restoration system for functional structures of proteins

蛋白质功能结构新修复系统的开发

基本信息

批准号：
15350103
负责人：
KUNUGI Shigeru
金额：
$ 9.6万
依托单位：
Kyoto Institute of Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-15350103/
关键词：
High pressure Protein Synthetic polymer Refolding Chaperone

项目摘要

The appropriate three dimensional structures are required for the high and specific functions of proteins. However, the general mechanism of converting sequential amino acid alignments into three dimensional structures is still ambiguous. Especially, irreversible denaturation or unfolding of proteins artificially formed by way of genetic engineering methods is a big problem.In this project, a new protein structure restoration or refolding system has been studied. There have been several attempts for protein structure restoration. They can be grouped into three categories : a-biochemical, b-chemical, and c-physicochemical. We have mainly introduced pressure as one the factor classed into c, and combined with chemical or biochemical methods.As the first stage, some biomimetic polymers such as an elastin-model peptide, stimulus-responsive synthetic polymers, have been investigated for their pressure-temperature responsive properties (structural stability and restoration). Then, structural stability and aggregation properties of several proteins, including phosphoglycerate kinase, a double-domain protein, kumamolisin, a single-domain protein, and insulin, amyloid-forming protein, have been studied. In addition, similarity and dissimilarity of proteins and biomimetic polymers including their hydro gels have been precisely compared.For the biochemical method, active-site fragments of molecular chaperone are targeted. Some active fragments have been prepared from cloned genes of DnaJ, ClpB and other molecular chaperones, and they were immobilized onto hydrophobic domain of phospholipid liposomes or micro-particles, to be utilized as the structure-restoration devices.

适当的三维结构是蛋白质的高级和特定功能所必需的。然而，将顺序氨基酸排列转化为三维结构的一般机制仍然不清楚。特别是，通过基因工程方法人工合成的蛋白质的不可逆变性或展开是一个大问题。在本项目中，研究了一种新的蛋白质结构恢复或重折叠系统。对于蛋白质结构的修复已经有过几次尝试。它们可以分为三类：a-生化、b-化学和c-物理化学。我们主要介绍了压力作为c类因素之一，并与化学或生化方法相结合。作为第一阶段，一些仿生聚合物，如弹性蛋白模型肽，刺激响应合成聚合物，已经研究了它们的压力-温度响应特性（结构稳定性和恢复）。然后，研究了几种蛋白质的结构稳定性和聚集特性，包括双结构域蛋白磷酸甘油酸激酶、单结构域蛋白kumamolisin和淀粉样蛋白胰岛素。此外，还精确地比较了蛋白质和仿生聚合物及其水凝胶的相似性和差异性。对于生化方法，分子伴侣的活性位点片段是靶向的。利用克隆的DnaJ、ClpB等分子伴侣基因制备了一些活性片段，并将其固定在磷脂脂质体或微颗粒的疏水结构域上，作为结构修复装置。