Method for protein design based on module units.

基于模块单元的蛋白质设计方法。

基本信息

批准号：
06558100
负责人：
GO Mitiko
金额：
$ 8.32万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

Our purpose is to establish method of protein design based on module units. Our aims are twofold : (1) design of stable mini-proteins by deletion of module from a native protein and analysis of its three-dimensional structure, and (2) design of mini-proteins having function of its mother protein, chemical synthesis and study of its function and three-dimensional structure. The followings are the main results obtained during the two-years research project.1) Design of a basic structure of mini-protein and evaluation of its conformational stability : We found that a mini-barnase designed by deletion of module M1 or M2 from barnase is a suitable candidate for designed mini-protein. We determined the amino acid replacements which were necessary to stabilize the mini-barnase by molecular dynamics calculation and evaluation of free energy change.2) The chemical synthesis of the designed mini-barnase : We synthesized the mini-protein by using the procedure developed by Hojo et al. Partially p … More rotected peptide thioesters corresponding to two peptides and partially protected peptide were prepared via a solid-phase method. For the introduction of residues at ten sites, ^<15>N-labeled amino acid derivatives were used. The thee peptide segments were successively condensed by the activation of thioester groups by silver ions to give a protected form of mini-barnase.3)Structure and function of the mini-barnases : On the basis of the chemical shift of the ^<15>N-labeled amino acids the mini-barnase without M2 was found to have a stable conformation similar with native barnase. The mini-barnase without M1 made a complex with module M1 and the complex had RNase activity.In conclusion, we were successful in design of mini-proteins by deletion of one module in the sense that the designed proteins were soluble and stable. Also, a method of chemical synthesis of mini-protein was developed. The determination of the three-dimensional structure of the mini-barnases by NMR measurements and development of methods for design of mini-barnase with enzymatic activity are left for further study. Less

我们的目的是建立基于模块单元的蛋白质设计方法。我们的目标是双重的：（1）通过从天然蛋白质中删除模块的稳定迷你蛋白质以及对其三维结构的分析，以及（2）在其母蛋白功能，化学合成和其功能研究和三维结构的功能的迷你蛋白质的设计。以下是在为期两年的研究项目中获得的主要结果。1）设计小型蛋白质的基本结构以及对其构象稳定性的评估：我们发现，通过从Barnase删除模块M1或M2设计的迷你 - 肉毒药酶是适合设计的Mini-Protein的候选者。我们确定了通过分子动力学计算和自由能变化评估稳定微型 - 肉毒药酶所必需的氨基酸替代品。2）设计的迷你 - 肉毒药的化学合成：我们使用Hojo等人开发的过程合成了微型蛋白。部分p…通过固相法制备了对两种肽的更旋转肽硫醇对应，并部分保护的肽。为了在十个位点引入残留物，使用了 ^<15> N标记的氨基酸衍生物。通过银离子激活硫酯基团的肽片段得到了微型 - 肉毒杆菌的受保护形式。3）微型肉毒药酶的结构和功能：根据 ^<15> n标记的氨基酸的化学移位，没有M2的小型氨基酸酶与MIN-BARNASE没有M2的稳定性与天然综合酶相似。没有M1的迷你 - 肉欲酶使模块M1变得复杂，并且复合物具有RNase活性。总之，我们通过删除一个模块成功地设计了迷你蛋白质，从某种意义上说，设计的蛋白质是固体且稳定的。同样，开发了一种化学合成方法。通过NMR测量结果确定微型 - 肉毒药酶的三维结构，并开发具有酶活性的迷你 - 肉毒药酶方法，以进行进一步研究。较少的