Elucidation of the mechanism of aggregation of disease-associated single point mutants of the murine prion protein on the basis of time-resolved 2D NMR spectroscopy in combination with complementary kinetic methods

已知朊病毒蛋白的聚集机制是复杂的。它是高度异质性的，因为多个低聚物以及纤维状结构被填充。了解疾病相关的朊病毒蛋白单体组装成高分子量聚合物的机制，以便能够开发定制的治疗方法是至关重要的。这需要在分子水平上详细跟踪聚集过程。动力学方法是特别适合在这方面所需的机械信息中所包含的动力学data.We研究了聚集的野生型鼠朊病毒蛋白使用动力学二维NMR光谱。通过以下单体浓度的降低，我们已经能够描绘的基础上的氨基酸特异性差异的聚集动力学的异质性。在拟议的项目范围内，我们的目标是扩展我们的动力学研究，并利用NMR的独特结构分辨率，这是使用其他方法无法实现的。除了测量单体浓度降低的动力学外，我们还特别计划使用2D NMR方法来表征淀粉样原纤维路径上不同低聚物群体的动力学。为此，我们将利用甲基同位素特异性标记方法与甲基TROSY NMR实验相结合。将使用原子力显微镜、SDS PAGE、荧光和吸收光谱法收集寡聚体和纤维状群体的补充动力学数据。最后，将从不同的实验动力学数据进行全局拟合，以获得与实验数据最相容的朊病毒蛋白聚集模型。然后，我们将表征疾病相关的单点突变体的朊病毒蛋白的聚集动力学，从而确定不同的突变影响的聚集子步骤。

The aggregation mechanism of the prion protein is known to be complex. It is highly heterogeneous since multiple oligomeric as well as fibrillar structures are being populated. It is essential to understand the disease-associated assembly of prion protein monomers into high-molecular-weight polymers in mechanistic terms in order to be able to develop tailored therapeutic approaches. This requires following the aggregation process in detail on the molecular level. Kinetic methods are especially suited in this respect as the desired mechanistic information is contained in the kinetic data.We have investigated the aggregation of the wildtype murine prion protein using kinetic two-dimensional NMR spectroscopy. By following the decrease in the monomer concentration we have been able to delineate heterogeneity in the aggregation kinetics on the basis of amino-acid-specific differences. Within the scope of the proposed project we aim at extending our kinetic studies and exploiting the unique structural resolution of NMR which cannot be attained using other methods. Apart from measuring the kinetics of the decrease in the monomer concentration we specifically plan to characterize the kinetics of distinct oligomeric populations on the path to amyloid-like fibrils using 2D NMR methods. In order to so, we will make use of methods for isotope-specific labelling of methyl groups in combination with methyl TROSY NMR experiments. Complementary kinetic data on oligomeric as well as fibrillar populations will be collected using atomic force microscopy, SDS PAGE, fluorescence and absorption spectroscopies. In the end, the kinetic data from the different experiments will be globally fitted in order to derive a model for the aggregation of the prion protein which is most compatible with the experimental data. We will then characterize the aggregation kinetics of disease-associated single point mutants of the prion protein and thereby identify the aggregation sub-steps affected by the different mutations.