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

我们建议研究的朊病毒蛋白的构象状态，在聚集的初始步骤，称为PrPC*，在温和的变性条件下，类似于以前用于产生感染性朊病毒和PrPSc样颗粒在体外。现代解决方案NMR技术将被应用于研究结构和动力学的PrPC* 合奏。特别是，通过多维异相NMR进行连续骨架分配后，我们将应用一整套NMR弛豫测量来检测与致病构象状态相关的运动和构象状态。站点特定的自旋标记和残留偶极耦合常数将描述残留结构的PrPC* 合奏。NMR研究将得到CD和荧光实验的支持。通过改变实验条件，我们将诱导朊病毒蛋白之间的瞬时接触，这将通过自旋标记NMR方法检测。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，弛豫方法将描述在天然条件下存在的不稳定的PrPC* 的量，并且自旋标记技术将用于测试朊病毒蛋白中促进PrPC -> PrPSc转变所需的局部解折叠的大呼吸运动。这项研究的系统性扩展到其他物种的朊病毒蛋白和致病性突变，最终将提供新的见解朊病毒疾病的机制和物种障碍与前所未有的细节。

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.