STRUCTURAL ANALYSIS OF SYNTHETIC PRIONS

在体内将细胞内的PrPC转变为另一种构象，即传染性PrPSc，这是PrPIC复制和病理过程中的病因学事件。直到最近，体外转化方案还未能从野生型PrP的重组来源或PrP的节段缺失变体中产生感染性PrP。最近，我们已经证明了体外转化的重组野生型小鼠PrP(89-230)可以引起一种历史上准确的神经退行性疾病，这种疾病是连续传播的。这一观察为推进对普恩的生物物理研究创造了几个机会。在体外，Prion显示出不寻常的菌株特征，由此推断，当传代到 野生型或转基因小鼠。我们建议通过纤维衍射、电子结晶学和分子模拟来研究这些人工合成的普里子的结构。目前，体外转化的效率仍然相当低。出于这个原因，我们将把最初的研究重点放在已经在老鼠身上传代的合成普鲁恩上。一旦体外转化过程的效率得到提高，我们计划进行更多的实验，直接从重组来源中鉴定合成的普恩病毒。我们最初的实验旨在分析形成淀粉样纤维和二维(2D)晶体的合成Prion，并将这些结果与自然产生的Prion的结果进行比较。实验数据将是 用于改进PrPSc的结构模型，该模型基于已知蛋白质或蛋白质结构域的结构。特别是，我们将把重点放在左手平行的β-螺旋作为一个基序，它可以解释由光学光谱数据所隐含的二级结构约束和由电子结晶学确定的空间约束。在历史上，光纤衍射结果对我们分析PrPSc的结构只贡献了低分辨率的数据。然而，我们相信，当结合建模和电子结晶学数据时，我们将能够从这些研究中提取更有意义的信息。因此，我们计划使用更先进的光纤对准方法来重新审视光纤衍射研究 基于同步加速器的X射线和电子衍射法。

The in vivo conversion of the cellular prion protein (PrPC) into an alternate conformer, the infectious prion (PrPSc), is the etiologic event in prion replication and pathology. Until recently, in vitro conversion protocols have failed to yield infectious prions from recombinant sources of wild-type PrP or segmental deletion variants of PrP. Recently, we have shown that in vitro converted, recombinant wild-type mouse PrP(89-230) can cause a historically accurate neurodegenerative disease that is serially transmissible. This observation creates several opportunities for advancing biophysical studies of prions. In vitro prions show unusual strain characteristics, and by inference structural properties, that are maintained when passaged in wild-type or transgenic mice. We propose to study the structure of these synthetic prions by fiber diffraction, electron crystallography, and molecular modeling. Currently, the in vitro conversion is still fairly inefficient. For this reason, we will focus our initial investigations on synthetic prions that have been passaged in mice. Once the efficiency of the in vitro conversion process is improved, we plan to carry out additional experiments to characterize synthetic prions directly from recombinant sources. Our initial experiments are designed to analyze synthetic prions that form amyloid fibers and two-dimensional (2D) crystals and to compare these results with those for naturally occurring prions. The experimental data will be used to refine structural models of PrPSc that are based on structures of known proteins or domains of proteins. In particular, we will focus on the left-handed parallel beta-helix as a motif that can account for the secondary structure constraints implied by optical spectroscopy data and spatial constraints determined by electron crystallography. Historically, fiber diffraction results have contributed only low-resolution data to our analysis of the structure of PrPSc. However, we believe that when coupled with modeling and electron crystallography data, we will be able to extract more meaningful information from these studies. Therefore, we plan to revisit fiber diffraction studies using more advanced methods of fiber alignment coupled with synchrotron-based X-ray and electron diffraction approaches.