Protein refolding and transient aggregate formation studied by very fast pressure

通过非常快的压力研究蛋白质重折叠和瞬时聚集体形成

• 批准号: 8269853
• 负责人: MARTIN GRUEBELE
• 金额: $ 26.02万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8269853
• 关键词: Affect; Aging; Complex; Creutzfeldt-Jakob Syndrome; Data; Disease; Drops; Employee Strikes; Energy Transfer; Environment; Equation; Equilibrium; Event; Fluorescence Resonance Energy Transfer; Goals; Heart; Heating; Hydrogen Bonding; Kinetics; Lead; Letters; Maps; Measures; Mesocricetus auratus; Methods; Modeling; Molecular; Mutation; Nature; Phase; PrP; Prions; Process; Proteins; Publishing; Relaxation; Reporting; Research; Resolution; Right-On; Sampling; Site; Solvents; Speed; Staging; Structure; Techniques; Temperature; Thermodynamics; Time; Work; density; design; insight; instrument; interest; lambda repressor; molecular dynamics; mutant; nanosecond; news; planetary Atmosphere; pressure; protein aggregation; protein folding; protein misfolding; prototype; public health relevance; research study; simulation; temperature jump; user-friendly

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.

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，比温度或溶剂添加剂更简单。我们在<1 nm的蛋白质样品上同时开发了一种新的压力跳跃，同时能够在<700纳秒中<700纳秒中的压力下降。该实验将用于研究叙利亚仓鼠Prion SHPRP的折叠和聚集的相互作用，该叙利亚仓鼠Prion SHPRP具有非常快的压力跳跃动力学相，这些相位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以及随后的天然状态形成如何与瞬态聚集竞争，最终可能导致原纤维形成。作为第二个压力跳跃项目，我们还详细研究了lambda阻遏物突变体的折叠，旨在改变蛋白质的包装和二级结构倾向。为了进行折叠研究，压降提供了一种替代的重折叠方法，该方法使二级结构少于温度跳跃实验。我们之所以选择Lambda阻遏物，是因为已经存在丰富的T-JUMP数据，并且T-JUMP和P-JUMP实验将很有趣。所有实验将通过折叠或折叠/聚集主方程动力学模型进行分析，该模型可与来自分子动力学仿真的马尔可夫动力学进行比较。我们与将进行相关模拟的舒尔特和潘德集团合作。目的是建立一个良好的低分辨率模型，以折叠或折叠/聚集能量的能量景观。 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。非常快速和大压力跳跃是研究Creutzfeldt-Jakob病蛋白模型的折叠与最早聚集步骤之间相互作用的新方法。