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

我们建议在温和变性的条件下，研究参与聚集初始步骤的Prion蛋白质的构象状态，称为PrPC*，类似于以前在体外用于产生感染性Prion和PrPSc样颗粒的构象。现代溶液核磁共振技术将被应用于研究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的主链指定之后，我们将应用一整套的核磁共振松弛测量来检测与致病构象状态相关的运动和构象状态。特定位置的自旋标记和剩余偶极耦合常数将描述PrPC*系综中的剩余结构。核磁共振研究将得到CD和荧光实验的支持。通过改变实验条件，我们将诱导蛋白质之间的瞬时接触，这将被自旋标记核磁共振方法检测到。这些实验将首次以高分辨率直接揭示普里子之间的接触。此外，松弛方法将描述在自然条件下存在的不稳定PrPC*的数量，自旋标记技术将被用于测试PrPC-&&PrPSc转变所需的促进局部展开的PrPC蛋白中的大呼吸运动。将这项研究系统地扩展到来自其他物种和具有致病突变的普恩蛋白，最终将以前所未有的细节为普恩疾病的机制和物种屏障提供新的见解。

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.