TOWARD A STRUCTURE OF PrPSc

PrPSc的不溶性阻碍了所有通过X射线结晶学或核磁共振光谱学来解决其结构的尝试。最近，我们发现了N端截短的PrPSc(PrP27-30)和编辑的小普里子(PrPSc106)的二维晶体。用电子结晶学分析2D晶体使我们能够映射PrP27-30和PrpSc106之间的差异。这些数据被用来约束PrPSc的结构模型。我们建议研究控制2D晶体生长的参数，以获得适合于低剂量低温电子结晶学的样品。这些晶体将被用来收集更高分辨率的数据，以生成三维 重建PrPSc的结构。我们还将寻求各种标记技术来定位分子的不同部分，以便在晶格上定位PrPSc的计算模型。实验数据将被用来改进PrPSc的结构模型，这些模型将基于已知蛋白质的结构或蛋白质的结构域。特别是，我们将把重点放在平行的β-螺旋作为一个基序，它可以解释FTIR光谱数据所暗示的二级结构约束和电子显微镜确定的空间约束。虽然纤维衍射结果在我们对PrPSc结构的分析中历来只贡献了低分辨率的结构数据，但我们相信，当结合模型和电子结晶学数据时，我们将能够从 这些研究。因此，我们希望使用更先进的光纤对准方法结合基于同步加速器的X射线和电子衍射方法来重新审视光纤衍射研究。此外，我们还想通过电子结晶学或纤维衍射学的方法来探索哪一种成熟的普里子菌株最适合进行结构分析。我们也有兴趣研究PrP序列截断，以支持PrPSc在转基因动物中的形成。这项工作将包括PrP 89-231，PrP 27-30的同系物，以及PrP106的变体，它们延长或缩小141-175的内部缺失，但仍然支持PrPSc的形成。

The insolubility of the scrapie prion protein (PrPSc) has frustrated all attempts to solve its structure by X-ray crystallography or NMR spectroscopy. Recently, we reported the discovery of two-dimensional (2D) crystals of the N-terminally truncated PrPSc (PrP 27-30) and a redacted miniprion (PrPSc106). Analyzing the 2D crystals by electron crystallography allowed us to map the differences between PrP 27-30 and PrpSc106. These data were used to constrain structural models of PrPSc. We propose to investigate the parameters that govern growth of the 2D crystals in order to obtain specimens suitable for low dose, cryo-electron crystallography. These crystals will be used to collect higher resolution data to generate a three-dimensional reconstruction of the structure of PrPSc. We will also pursue various labeling techniques to localize different parts of the molecule in order to position computational models of PrPSc on the crystal lattice. The experimental data will be used to refine structural models of PrPSc that will be based on structures of known proteins or domains of proteins. In particular, we will focus on the parallel Beta-helix as a motif that can account for the secondary structure constraints implied by FTIR spectroscopy data and spatial constraints determined by electron microscopy. While fiber diffraction results have historically contributed only low-resolution structural data to our analysis of the structure of PrPSc, we believe that when coupled with the models and electron crystallography data, we will be able to extract more meaningful information out of these studies. Therefore, we want to revisit fiber diffraction studies using more advanced methods of fiber alignment coupled with synchrotron-based X-ray and electron diffraction approaches. Furthermore, we want to explore which of the well-established prion strains are most suitable for structural analysis by either electron crystallography or by fiber diffraction. We are also interested in studying PrP sequence truncations that can support PrPSc formation in transgenic animals. This work will include PrP 89-231, a homolog of PrP 27 - 30, and variants of PrP106 that extend or shrink the internal deletion from 141 - 175 yet still support PrPSc formation.