Protein refolding and transient aggregate formation studied by very fast pressure

描述（由申请人提供）：蛋白质折叠和聚集是不断相互作用的过程，有时会导致疾病。蛋白质折叠和蛋白质聚集都对压力敏感，这是一个比温度或溶剂添加剂更简单的变量。我们开发了一种新的压力跳跃，同时能够在< 700纳秒内对< 1 nM的蛋白质样品产生> 2500 atm的压降。该实验将用于研究叙利亚仓鼠朊病毒ShPrP的折叠和聚集的相互作用，已知该朊病毒具有非常快的压力跳跃动力学阶段，无法通过常规仪器解决。压力和浓度依赖性研究将揭示早期折叠中间体的形成和随后的天然状态的形成如何与最终可能导致形成原纤维的瞬时聚集竞争。作为第二个压力跳跃项目，我们还详细研究了λ阻遏突变体的折叠，这些突变体旨在改变蛋白质的包装和二级结构倾向。对于折叠的研究，压降提供了一种替代的重折叠方法，扰动二级结构比温度跳跃实验。我们选择λ阻遏物是因为已经存在丰富的T-跳跃数据，并且T-跳跃和P-跳跃实验将是有趣的比较。所有实验将使用折叠或折叠/聚集主方程动力学模型进行分析，该模型可用于与分子动力学模拟得出的马尔可夫动力学进行比较。我们与Schulten和Pande团队合作，他们将进行相关的模拟。我们的目标是建立一个良好的低分辨率模型的折叠或折叠/聚集能量景观的这些蛋白质。 公共卫生相关性：许多疾病是由蛋白质折叠和聚集之间的竞争引起的。非常快和大的压力跳跃是研究克雅氏病蛋白模型的折叠和最早聚集步骤之间相互作用的新方法。

DESCRIPTION (provided by applicant): Protein folding and aggregation are processes that constantly interact, sometimes leading to disease. Both protein folding and protein aggregation are sensitive to pressure, a thermodynamically simpler variable than temperature or solvent additives. We develop a new pressure jump simultaneously capable of > 2500 atm pressure drops in < 700 nanoseconds on < 1 nM of protein sample. The experiment will be used to study the interplay of folding and aggregation of the Syrian hamster prion ShPrP, which is known to have very fast pressure-jump kinetic phases that could not be resolved by conventional instruments. Pressure- and concentration-dependent studies will reveal how formation of early folding intermediates and subsequent formation of the native state compete with transient aggregation that could eventually lead to formation of protofibrils. As a second pressure jump project, we also study in detail the folding of lambda repressor mutants designed to alter the packing and secondary structure propensity of the protein. For folding studies, the pressure drop provides an alternative refolding method that perturbs secondary structure less than temperature jump experiments. We picked lambda repressor because rich T-jump data already exist, and T-jump and P-jump experiments will be interesting to compare. All experiments will be analyzed with a folding or folding/aggregation master equation kinetics model that is useful for comparison with Markov dynamics derived from molecular dynamics simulation. We collaborate with the Schulten and Pande group who will carry out relevant simulations. The goal is to build a good low-resolution model of the folding or folding/aggregation energy landscape of these proteins. PUBLIC HEALTH RELEVANCE: Many diseases are caused by the competition between folding and aggregation of proteins. Very fast and large pressure jumps are a new way of studying the interplay between folding and the earliest aggregation step of a model of the Creutzfeldt-Jakob disease protein.