TOWARD A STRUCTURE OF PrPSc

羊瘙痒病朊病毒蛋白（PrPSc）的不溶性使所有试图通过X射线晶体学或核磁共振光谱学来解决其结构的尝试都失败了。最近，我们报道了发现的二维（2D）晶体的N-末端截短的PrPSc（PrP 27-30）和编辑miniprion（PrPSc 106）。通过电子晶体学分析2D晶体使我们能够绘制PrP 27-30和PrpSc 106之间的差异。这些数据用于约束PrPSc的结构模型。我们建议调查的参数，以获得适合于低剂量，低温电子晶体学的标本的2D晶体的生长。这些晶体将用于收集更高分辨率的数据， PrPSc结构的重建。我们还将采用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。实验数据将用于完善PrPSc的结构模型，该模型将基于已知蛋白质或蛋白质结构域的结构。特别是，我们将专注于平行β-螺旋作为一个主题，可以解释的FTIR光谱数据和电子显微镜确定的空间限制所暗示的二级结构的约束。虽然纤维衍射结果在历史上只为我们对PrPSc结构的分析提供了低分辨率的结构数据，但我们相信，当与模型和电子晶体学数据相结合时，我们将能够从PrPSc结构中提取更有意义的信息。 这些研究。因此，我们希望重新审视光纤衍射研究，使用更先进的方法，光纤对准耦合同步加速器为基础的X射线和电子衍射方法。此外，我们想探索哪一种公认的朊病毒株最适合通过电子晶体学或纤维衍射进行结构分析。我们也有兴趣研究PrP序列截短，可以支持PrPSc形成转基因动物。这项工作将包括PrP 89-231，PrP 27 - 30的同源物，以及PrP 106的变体，这些变体从141 - 175延伸或收缩内部缺失，但仍然支持PrPSc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.