Development of design method to get soluble protein

开发获得可溶性蛋白质的设计方法

基本信息

批准号：
08559009
负责人：
GO Mitiko
金额：
$ 6.59万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08559009/
关键词：
making protein soluble barnase mini-protein reverse transcriptase amino acid replacement domain fusion protein evolution module

项目摘要

In order to know structure and function of gigantic protein and protein complex, a method to cut and take out stable partial structure of protein and a method to make the partial structure soluble are needed. Our purpose is to develop a design method to take out partial structure of protein in a soluble and stable from. We intended further to verify that the designed partial structure forms soluble and stable structure in solvent. In process of molecular evolution, a fusion of domain or module occurred frequently. We showed that in reverse transcriptase, on the occasion of RNaseH domain fusion, at least 4 amino acid replacements occurred in adopting to the atomic interactions at domain contact. We applied the same method to make a partial protein structure soluble. Previously we found a barnase-like domain in RNA polymerase. We planed to cut out the barnase-like domain from RNA polymerase and designed a soluble form of isolated barnase-like domain. Then we did a chemical synthesis of the sequence. For getting soluble barnase-like domain, we established a method to replace amino acid residues using an evolutionary replacement pattern of amino acid in domain fusion. This method is applicable for the proteins whose three-dimensional structures are unknown. Also, we designed a mini-barnase by removing a module from barnasr, performed molecular dynamics and evaluated its solubility and stability by free energy calculation. Mini-barnase lacking 26 amino acid residues was chemically synthesized. We measured CD and NMR of designed mini-barnase. It was dissolved into water and took a stable structure similar to the conformation of a barnase except the excised region. Through this research, we developed a method that is useful in cutting and taking out a part of protein in soluble and stable form.

为了了解巨型蛋白质和蛋白质复合物的结构和功能，需要一种剪切和消除稳定的蛋白质局部结构的方法，以及一种使部分结构可溶的方法。我们的目的是开发一种设计方法，以在可溶性和稳定的情况下取出蛋白质的部分结构。我们打算进一步验证设计的部分结构在溶剂中形成可溶性和稳定的结构。在分子进化过程中，域或模块的融合经常发生。我们表明，在逆转录酶中，在RNASEH结构融合的情况下，在域接触处采用原子相互作用时，至少发生了至少4个氨基酸替代。我们应用了相同的方法来使部分蛋白质结构溶解。以前，我们在RNA聚合酶中发现了一个类似巴尔米酶的结构域。我们计划从RNA聚合酶中切出类似于巴尔纳酶的结构域，并设计了一种可溶性的孤立的barnase样结构域。然后，我们进行了序列的化学合成。为了获得可溶性barnase样结构域，我们建立了一种使用域融合中氨基酸进化替代模式替代氨基酸残基的方法。该方法适用于其三维结构未知的蛋白质。此外，我们通过从Barnasr中删除一个模块，进行了分子动力学，并通过自由能计算评估了其溶解度和稳定性，从而设计了一个迷你 - 狂欢酶。缺乏26个氨基酸残基的迷你 - 狂欢酶化学合成。我们测量了设计的迷你 - 肉毒药酶的CD和NMR。它被溶解在水中，并采用了类似于切除区域除外的barnase构象的稳定结构。通过这项研究，我们开发了一种可在可溶性和稳定形式切割和取出一部分蛋白质的方法。