Development of new restoration system for functional structures of proteins

蛋白质的高度和特定功能需要合适的三维结构。然而，将氨基酸序列比对转换为三维结构的一般机制仍然不明确。尤其是通过基因工程方法人工形成的蛋白质的不可逆变性或去折叠是一个很大的问题。本项目研究了一种新的蛋白质结构恢复或复性系统。已经有过几次恢复蛋白质结构的尝试。它们可以分为三类：a-生化、b-化学和c-物理化学。我们主要引入压力作为c的一个因素，并结合化学或生化方法。作为第一阶段，我们研究了一些仿生聚合物，如弹性蛋白模型肽，刺激响应型合成聚合物，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和淀粉样蛋白形成蛋白胰岛素。此外，还比较了蛋白质和仿生聚合物及其水凝胶的异同。对于生化方法，以分子伴侣的活性部位片段为目标。从克隆的DNAJ、ClpB等分子伴侣基因中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域上，作为结构修复装置。

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.