Elucidatiop of Tangle Formation Mechanism Based on 3D-structural Analysis of Tau Protein

1）微管结合区的X射线晶体结构分析为了阐明tau蛋白的三维结构，我们尝试了tau蛋白及其片段的各种结晶。因此，我们成功地结晶tau微管结合结构域（MBD）作为GST融合蛋白，这是通过基因表达从大肠杆菌中制备的重组体。晶体数据为：四氢呋喃，空间群P4_32_12，a=B=92.24，c=57.68。X-射线衍射数据高达2 μ m的分辨率，收集同步加速器的春天8。利用GST的三维结构，通过分子置换法求解了晶体结构。经过多次结构优化，目前的R因子为0.258。虽然MBDs形成二聚体结构，但它们表现出较大的构象灵活性，并且它们的原子位置无法确定。这与GST的刚性3D结构形成对比。由于MBD构建了tau PHF的核心结构，因此本研究结果表明，tau PHF的构象与其蛋白质结构之间存在密切关系。 ...更多信息 MBD和PHE形成的相对灵活性。2）MBDMBD的溶液结构分析表明，MBDMBD由三个或四个重复序列组成，每个重复序列（R1 -R4）由31或32个氨基酸残基组成。为了阐明每个重复肽的构象灵活性，通过NMR和模型建立相结合的方法分析了R1 -R4在TEE溶液中的溶液结构。因此，R1、R2和R4肽段的结构均为α-螺旋结构，氨基酸残基呈两亲性分布。而R3则同时具有两亲性的α-螺旋和伸展螺旋结构。3）MBD相互作用的研究和重复结构依赖的PHF形成模型通过ThS荧光、光散射和表面等离子体共振分析以及电子显微镜研究了相邻MBD之间的缔合作用。在此基础上，我们提出了第一个由重复结构控制的4 RMBD逐步成丝模型。该模型将有助于考虑合理的方法来防止AD Less中的tau沉积

1)X-ray crystal structure analysis of microtubule-binding domainTo elucidate 3D structure of tau protein, we attempted various crystallizations of tau and its fragments. Consequently, we succeeded in the crystallization of tau microtubule-binding domain (MBD) as GST fusion protein, which were prepared as recombinant by gene expression from E.coli. The crystal data are : tetragonal, space group P4_32_12, a=b=92.24Å, c=57.68Å. X-ray diffraction data up to 2Å resolution were collected by synchrotron of Spring 8. The crystal structure was solved by the molecular replacement method using the 3D structure of GST. After several structure refinement, the present R factor is 0.258. Although the MBDs formed dimeric structure, they showed large conformational flexibility and their atomic positions were not determined. This is in contrast with the rigid 3D structure of GST. Since the MBD constructs the core structure of tau PHF, the present result indicates the close relationship between the confo … More rmational flexibility of MBD and PHE formation. We are now in progress in analyzing the crystal structure more accurately.2)Analyses of solution structure of MBDMBD is composed of three-or four-repeated structure, where each repeat peptide (named R1 -R4) consists of 31 or 32 amino acid residues. To elucidate the conformational flexibility of each repeat peptide, the solution structures of R1 -R4 in TEE solution were analyzed by the combination of NMR and model building. Consequently, the the structures of R1, R2 and R4 peptides showed α-helical structure of amphipathic distribution of respective amino acid residues. In contrast, R3 took both of extended and α-helical structures of amphipathic behavior. This result suggest the molecular association of MBD through alternative hydrophilic and hydrophobic interactions.3)Investigation of MBD interactions and model of repeat-structure-dependent PHF formationThe association of neighboring MBDs were investigated by ThS fluorescence, light scattering and surface plasmon resonance analyses, and electromicroscopy. On the basis of these results, we proposed the first model of repeat structure-controlled step-by-step filament formation of 4RMBD. This model would be helpful upon considering rational approaches to prevent tau deposition in AD Less