Method for protein design based on module units.

我们的目的是建立基于模块单元的蛋白质设计方法。我们的目标有两个：(1)通过从天然蛋白质中删除模块并分析其三维结构来设计稳定的微型蛋白质；(2)设计具有母体蛋白质功能的微型蛋白质，化学合成及其功能和三维结构的研究。1)迷你蛋白基本结构的设计及其构象稳定性的评价：我们发现，通过从棒状酶中删除模块M1或M2而设计的迷你藤本酶是设计的迷你蛋白的合适候选者。通过分子动力学计算和自由能变化评价，确定了稳定迷你藤壶酶所需的氨基酸替换。2)设计的迷你藤壶酶的化学合成：采用Hojo等人开发的方法合成了迷你蛋白。部分p…用固相法制备了更多对应于两种多肽和部分保护多肽的受保护多肽硫酯。为了在十个位置引入残基，使用了^&lt；15&gt；N标记的氨基酸衍生物。3)迷你藤壶酶的结构和功能：根据~&lt；15&gt；N标记氨基酸的化学位移，发现不含M2的迷你藤壶酶具有与天然藤壶酶相似的稳定构象。不含M1的迷你Barnase与模块M1形成复合体，该复合体具有核糖核酸酶活性，通过删除一个模块，我们成功地设计出了具有可溶性和稳定性的微型蛋白质。此外，还建立了一种微量蛋白质的化学合成方法。通过核磁共振测量确定迷你藤壶酶的三维结构和开发具有酶活性的迷你藤壶酶的设计方法还有待进一步研究。较少

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