TOWARD A STRUCTURE OF PrPSc

走向 PrPSc 的结构

• 批准号: 6742807
• 负责人: FRED E COHEN
• 金额: $ 21万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-01-01 至 2008-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6742807
• 关键词: X ray crystallography; computational biology; electron crystallography; electron microscopy; infrared spectrometry; physical model; prions; protein sequence; protein structure; scrapie; structural biology

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.

痒病朊病毒蛋白 (PrPSc) 的不溶性使得所有通过 X 射线晶体学或核磁共振波谱解析其结构的尝试都失败了。最近，我们报道了 N 端截短的 PrPSc (PrP 27-30) 和编辑的小朊病毒 (PrPSc106) 的二维 (2D) 晶体的发现。通过电子晶体学分析 2D 晶体，我们能够绘制出 PrP 27-30 和 PrpSc106 之间的差异。这些数据用于约束 PrPSc 的结构模型。我们建议研究控制二维晶体生长的参数，以获得适合低剂量冷冻电子晶体学的样本。这些晶体将用于收集更高分辨率的数据以生成三维 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，以便将 PrPSc 的计算模型定位在晶格上。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，我们将重点关注平行 Beta 螺旋作为基序，它可以解释 FTIR 光谱数据暗示的二级结构约束和电子显微镜确定的空间约束。虽然纤维衍射结果历来只为我们对 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，我们将能够从其中提取更有意义的信息。 这些研究。因此，我们希望使用更先进的光纤对准方法以及基于同步加速器的 X 射线和电子衍射方法来重新审视光纤衍射研究。此外，我们想探索哪些成熟的朊病毒菌株最适合通过电子晶体学或纤维衍射进行结构分析。我们还对研究可支持转基因动物中 PrPSc 形成的 PrP 序列截断感兴趣。这项工作将包括 PrP 89-231（PrP 27 - 30 的同源物）以及 PrP106 的变体，这些变体将内部缺失从 141 - 175 延伸或缩小，但仍支持 PrPSc 的形成。