Protein refolding and transient aggregate formation studied by very fast pressure

描述(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很敏感，压力是一个比温度或溶剂添加剂更简单的热力学变量。我们开发了一种新的压力跳跃，能够同时在&lt；1纳米蛋白质样品上同时&gt；2500 atm压力下降&lt；700纳秒。这项实验将被用来研究叙利亚仓鼠PrP蛋白的折叠和聚集的相互作用，众所周知，ShPrP具有传统仪器无法分辨的非常快的压力跳跃动力学阶段。压力和浓度依赖的研究将揭示早期折叠中间产物的形成和随后的本征状态的形成如何与最终可能导致原纤维形成的瞬时聚集竞争。作为第二个压力跃升项目，我们还详细研究了Lambda阻遏突变体的折叠，该突变体旨在改变蛋白质的堆积和二级结构倾向。对于折叠研究，压力降提供了一种替代的折叠方法，与温度跳跃实验相比，该方法对二级结构的干扰较小。我们选择了波长抑制因子，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。所有实验都将使用折叠或折叠/聚集主方程动力学模型进行分析，该模型可用于与来自分子动力学模拟的马尔可夫动力学进行比较。我们与Schulten和Pande小组合作，他们将进行相关的模拟。我们的目标是建立一个很好的低分辨率模型来描述这些蛋白质的折叠或折叠/聚集的能量格局。 与公共卫生相关：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.