Preventing Protein Aggregation by Controlling Unfolded State Dynamics

描述(申请人提供)：未折叠的蛋白质是高度复杂的对象，包含天然和非天然的相互作用，但仍然能够扩散地重新配置。PI实验室的最新结果表明，折叠条件下的未折叠蛋白质显示出广泛的分子内扩散系数，跨越三个数量级。分子内扩散和聚集倾向之间存在着新的相关性，聚集倾向的蛋白质占据了这个动态范围的中间。为了了解聚集和未折叠蛋白质动力学之间的关系，该项目将测量各种易于聚集的序列中的分子内扩散，以及扩散如何随着突变而变化。为了将这种关系应用于药物设计，将观察小分子聚集抑制剂对扩散的影响。等电点采用了半胱氨酸猝灭色氨酸三重态的新技术，该技术是用一台纳秒分辨率的光学仪器测量的。分子内扩散系数可以用Szabo，Schulten和Schulten的理论从这些测量的速率中提取出来，该理论要求序列中色氨酸和半胱氨酸之间的平衡距离的概率分布。这个项目的一个关键方面是通过全原子分子动力学或PI开发的聚合物模型对概率分布进行计算建模。阿尔茨海默氏症和α-突触核蛋白将在平衡状态下进行测量，氢酶成熟蛋白和各种哺乳动物蛋白将在一种新型的微流控混合器中进行测量，该混合器可以快速稀释变性剂 在~250μS。

DESCRIPTION (provided by applicant): Unfolded proteins are highly complex objects, containing native and non-native interactions but still remain able to reconfigure diffusively. Recent results from the PI's lab show that unfolded proteins under folding conditions show a wide range of intramolecular diffusion coefficients, spanning three orders of magnitude. There is an emerging correlation between intramolecular diffusion and aggregation propensity, with aggregation-prone proteins occupying the middle of this dynamic range. To understand the relationship between aggregation and unfolded protein dynamics, this project will measure intramolecular diffusion in a variety of sequences prone to aggregation and how diffusion changes with mutation. To apply this relationship to drug design, the effect of small molecule aggregation inhibitors on diffusion will be observed. The PI uses the novel technique of quenching of the triplet state of tryptophan by cysteine, which is measured with an optical instrument with nanosecond resolution. Intramolecular diffusion coefficients can be extracted from these measured rates using a theory by Szabo, Schulten and Schulten which requires a probability distribution of equilibrium distances between the tryptophan and cysteine in the sequence. A crucial aspect of this project is the computational modeling of the probability distribution by either all-atom molecular dynamics or a polymeric model developed by the PI. Alzheimer's Aß and α-synuclein will be measured in equilibrium, and hydrogenase maturation protein and various mammalian prion proteins will be measured in a novel microfluidic mixer that rapidly dilutes denaturant in ~250 μs.