Development of Novel Methods for Structural Analyses of Proteins

该小组成员致力于开发分析蛋白质结构及其相互作用的新方法。发展了一种利用固体核磁共振研究膜中蛋白质等无序固体蛋白质三维结构的新方法。通过溶液核磁共振的基本方法学研究，确定了溶液中转录因子等一系列蛋白质结构。基于一种新的高分子量糖蛋白的标记方法，建立了一种新的方法。结合核磁共振和标记法可以获得有用的免疫球蛋白G复合体的结构数据。基于硫代酯法，发展了一种用于结构研究的有效的蛋白质化学制备方法，包括磷蛋白和糖蛋白等结合蛋白。建立了稳定同位素标记的核酸中氨基酸和碱基的合成方法。利用化学合成的标记DNA，用核磁共振方法确定了蛋白质-DNA复合体的结构。发展了一种解决超大分子晶体结构的新技术，使我们能够将水稻矮缩病毒晶体的X射线衍射上限从20A提高到原子分辨率。为了分析生物大分子与生物大分子聚集态结构的相互作用，研制了用于晶体结构分析的X射线小角散射系统、中子散射数据采集方法和探测器。提出了一种基于核磁共振数据生成蛋白质结构和蛋白质-DNA复合体结构的新算法。

The members of this group focused their efforts on the development of novel methods for the analyses of protein structures and of their interactions. A new method was developed to elucidate the three-dimensional structure of unordered solid-state proteins such as proteins in a membrane by employing the solid-state NMR. A series of protein structures such as transcription factors in solution were determined by NMR through the basic methodological studies of solution NMR. A new method was developed based on a novel labeling method of a high molecular weight glycoprotein. Useful structural data of immunoglobulin G complex could be obtained by using the combination of the labeling method and NMR. An efficient method for the chemical preparation of proteins, including conjugated proteins such as phosphoproteins and glycoproteins, for structural studies was developed based on a thioester method. Synthetic method of stable-isotope labeled amino acids and bases in nucleic acids were established. Using a chemically synthesized labeled DNA, the structure of protein-DNA complex was determined by NMR. A novel technique to solve the crystal structures of super-macromolecules was developed, enabling us to increase the upper resolution limit of x-ray diffraction of Rice Dwarf virus crystals from 20A up to atomic resolution. In order to analyze the interaction between bio-macromolecules and aggregated structure of biomacromolecules, a system for x-ray small angle scattering, and a data collection method and a detector for neutron scattering for crystal structure analysis were developed. A new efficient algorithm to generate the structures of proteins and protein-DNA complexes was developed based on the NMR data.