Struktur und Dynamik von Konformeren des Prionproteins im Verlauf der pathogenen Konformationsänderung PrPC -> PrPSc

我们建议在轻度变性条件下研究参与聚集初始步骤的朊病毒蛋白（称为 PrPC*）的构象状态，类似于之前用于在体外生成传染性朊病毒和 PrPSc 样颗粒的条件。现代溶液核磁共振技术将用于研究 PrPC* 系综的结构和动力学。特别是，在通过多维异核核磁共振进行连续主链分配后，我们将应用全套核磁共振弛豫测量来检测与致病构象状态相关的运动和构象状态。位点特异性自旋标记和残余偶极耦合常数将描述 PrPC* 系综中的残余结构。核磁共振研究将得到圆二色和荧光实验的支持。通过修改实验条件，我们将诱导朊病毒蛋白之间的瞬时接触，这将通过自旋标记核磁共振方法进行检测。这些实验将首次以高分辨率直接揭示朊病毒间的接触。此外，松弛方法将描述天然条件下存在的不稳定 PrPC* 的量，并且自旋标记技术将用于测试朊病毒蛋白中的大呼吸运动，促进 PrPC -> PrPSc 转变所需的局部解折叠。这项研究系统地扩展到其他物种的具有致病性突变的朊病毒蛋白，最终将为朊病毒疾病的机制和物种屏障提供前所未有的细节新见解。

We propose to study conformational states of the prion protein involved in the initial steps in aggregation, termed PrPC*, under mildly denaturing conditions, similar to those previously used to generate infectious prions and PrPSc-like particles in vitro. Modern solution NMR-techniques will be applied to study structure and dynamics of the PrPC* ensemble. In particular, after sequential backbone-assignment by multidimensional heteronuclear NMR, we will apply a full set of NMR-relaxation measurements to detect motions and conformational states relevant to the pathogenic conformational state. Site-specific spin-labelling and residual dipolar coupling constants will describe residual structure in PrPC* ensemble. NMR studies will be supported by CD and fluorescence experiments. By modification of the experimental conditions we will induce transient contacts between prion proteins, which will be detected by spin-label NMR-methods. These experiments will for the first time directly reveal inter-prion contacts at high resolution. Further, relaxation methods will describe the amount of destabilized PrPC* present under native conditions, and spin-label techniques will be used to test for large breathing-motion in prion proteins promoting local unfolding required for the PrPC -> PrPSc transition. The systematic extension of this study to prion proteins form other species and with pathogenic mutations will ultimately provide novel insights into the mechanism of prion diseases and the species barrier with unpreceded detail.