激光光镊技术对α-synuclein蛋白折叠与聚集动力学研究

Alpha-synuclein（α-syn）is the most important composition of Lewy bodies in familial Parkinsonism. The misfolding and aggregation of α-syn protein are strongly linked to Parkinson’s disease (PD). So far, folding and aggregation kinetics of α-syn protein are not clear. It is because that the intermediates formed during the aggregations are typically transient and often coexisting with many other structures which makes it is difficult to study using conventional techniques such as crystallization. Focusing on the problems mentioned above, we will study the folding and aggregation kinetics of α-syn in single-molecule resolution by using the optical tweezers. Firstly, we will improve the detection precision and performance of our optical tweezers system. Then, we will study the effects of temperature, pH value, stress and other factors on the kinetics of α-syn aggregation at high resolution using single-molecule force spectroscopy, and also study the conformational transitions of several mutations (A53T, A30P) of α-syn and their inferences in the aggregations. Thirdly, we will also focus on the intermediates of α-syn oligomers exist in the conformational transition and their dynamics during the aggregations. And finally, find the mechanisms of the molecular chaperones in regulating the aggregation of α-syn. This project is expected to reveal the cause and kinetics of α-syn protein aggregations which are great significance to understanding the pathogenesis of Parkinson’s disease.

Alpha-synuclein（α-syn）是帕金森症的特征性标志物路易小体的重要组成成分，其错误折叠、聚集与帕金森症发生发展密切相关。α-syn蛋白的折叠和聚集动力学机制目前尚不明了，这主要归因于，α-syn蛋白聚集过程中产生的寡聚体中间态，存在时间很短且与其他结构同时出现，难以采用结晶等常规技术研究。针对α-syn蛋白聚集动力学研究中的上述问题，本项目拟在前期构建光镊系统的研究基础上，优化提高其探测精度和可控性能；利用光镊原位、实时探测的特性，采用单分子力谱，在单分子水平上研究温度、pH值、压力等因素对α-syn蛋白聚集动力学，以及A53T和A30P突变对α-syn蛋白构象和聚集动力学的影响； 研究聚集过程中α-syn蛋白寡聚体构象变化的中间态及其动力学特性；探索分子伴侣调控α-syn蛋白聚集行为的作用机制。本项目有望揭示α-syn蛋白的聚集原因和聚集动力学，深化人们对帕金森症发病机制的认识。

Alpha-synuclein是帕金森症的特征性标志物路易小体的重要组成成分，其错误折叠、聚集与帕金森症发生发展密切相关。α-syn蛋白的折叠和聚集动力学机制目前尚不明了。为了解决α-syn蛋白折叠动力学的问题，本项目搭建了一套高时间分辨率和高空间分辨率的双光镊单分子测量系统，其位移测量精度能到纳米量级。利用该系统，我们研究了KIF11马达蛋白在微管上行走的动力学特性，成功的测量到了KIF11马达蛋白约8nm的行走步幅。研究了单个α-syn蛋白折叠特性，和A53T与A29P突变对α-syn蛋白构象和聚集动力学的影响。并且研究了不同的环境如pH值对于其构象变化的影响。这些研究将有助于揭示α-syn蛋白聚集的原因以及帮助找到解决的方法。