Method for protein design based on module units.

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并分析其三维结构来设计稳定的微型蛋白质，和（2）设计具有其母蛋白质功能的微型蛋白质，化学合成并研究其功能和三维结构。1）微型蛋白基本结构的设计及其构象稳定性的评价：我们发现通过缺失芽孢杆菌RNA酶的M1或M2模块设计的微型芽孢杆菌RNA酶是设计微型蛋白的合适候选者。通过分子动力学计算和自由能变化的评价，确定了稳定该酶所必需的氨基酸替换。2）设计的mini-barnase的化学合成：采用Hojo等人开发的方法合成了mini-barnase蛋白，部分合成了mini-barnase蛋白。 关于我们 通过固相法制备对应于两种肽和部分保护的肽的保护的肽硫酯。为了在10个位点引入残基，使用了<15>N-标记的氨基酸衍生物。3）微型芽孢杆菌RNA酶的结构与功能：根据~（13）<15>N-标记氨基酸的化学位移，发现不含M_2的微型芽孢杆菌RNA酶具有与天然芽孢杆菌RNA酶相似的稳定构象。不含M1的微型芽孢杆菌RNA酶与M1模块形成复合物，该复合物具有RNase活性。此外，还发展了一种化学合成微蛋白的方法。通过核磁共振测量确定微型芽孢杆菌RNA酶的三维结构以及开发具有酶活性的微型芽孢杆菌RNA酶的设计方法还有待进一步研究。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