Collaborative Research: The Thermodynamics of Protein Separations

合作研究：蛋白质分离的热力学

• 批准号: 0078491
• 负责人: Michael Paulaitis
• 金额: $ 29.95万
• 依托单位: Johns Hopkins University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-02-15 至 2005-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0078491&HistoricalAwards=false
• 关键词: Collaborative; Research; Thermodynamics; Protein; Separations

ABSTRACTCTS-0078491Michael PaulaitisJohns Hopkins U.The objective of the proposed research is to develop quantitative tools for the analysis and design of protein separation processes. These tools will be compared to those now used routinely to design conventional separations in the chemical process industries. This development is based on combining theory and experiment to enable a new generation of thermodynamic models capable of describing mixtures of proteins.The approach builds on the paradigm that has so far emerged from our work, namely that protein thermodynamic properties are dominated by specific biological interactions in the form of geometric complementarity. It is believed that these specific interactions can be understood and accounted for in terms of electrostatic, dispersion, and hydrophobic interactions. This proposal is centered on extending our understanding of these interactions and finding optimal ways in which to manipulate and exploit them in designing effective separation strategies. Specifically, we will:1. complete our, model formulation to allow efficient mechanistic calculation of protein-protein interactions for both like and unlike protein pairs; 2. measure the model interactions between like and unlike protein pairs, and correlate results with measure separation performance. 3. use interactions between unlike protein pairs as the basis for developing strategies for optimizing protein separation trains.The molecular theories will be based on recently developed computational methods to account for interactions, and will be combined into an accurate potential of mean force (PMF) describing the interactions between both like and unlike proteins. This PMF is the foundation for the foundation for the description of both the physical properties of the protein solution thermodynamic function that determine the phase behavior. Several computational and molecular simulation methods will be explored for calculating of the phase behavior of the protein solutions. Our working hypothesis is that is the relative magnitudes of like vs, unlike protein interactions that determine how a particular separation can best be accomplished.An array of experimental measurements will complement and guide theory. The experiments first involve characterization of structural and functional protein properties using light scattering and small angle neutron scattering (SANS). Next, measurements of thermodynamic and physical properties and mixtures of proteins will be made to test the validity of the thermodynamic descriptions. One thermodynamic variable of particular interest will be pressure. Specifically the investigator will investigate the effect of pressure on protein stability and how protein folding can be manipulated with pressure to control protein separations and purification. Finally, separation experiments will be conducted to evaluate the predictions of performance based on the thermodynamic models. These measurements and calculations will guide efforts to find conditions under which adverse interactions impair separation effectiveness, and to design and test suitable remedies. Several proteins and protein mixtures will be examined, including eglin C and staphylococcal nuclease.This combination of experimental and theoretical tools will identify the properties of proteins important in the design of separation processes, and will allow separation performance to be predicted on the basis of protein properties. The project is part of a group proposal with investigators at the University of Delaware (0078844).

本研究的目的是开发定量工具，用于蛋白质分离过程的分析和设计。 这些工具将比较那些现在经常用来设计传统的分离在化学加工工业。 这一发展是基于理论和实验相结合，使新一代的热力学模型能够描述蛋白质的混合物。该方法建立在迄今为止从我们的工作中出现的范式之上，即蛋白质的热力学性质由特定的生物相互作用以几何互补的形式主导。 据信，这些特定的相互作用可以理解和占静电，分散和疏水相互作用。 这项建议的核心是扩展我们对这些相互作用的理解，并找到最佳的方法来操纵和利用它们来设计有效的分离策略。 具体来说，我们将：1。完成我们的模型制定，以允许有效的机械计算蛋白质-蛋白质相互作用的相似和不相似的蛋白质对; 2. 测量相似和不相似蛋白质之间的模型相互作用 配对，并将结果与测量分离性能相关联。 3. 使用不同蛋白质对之间的相互作用作为基础， 分子理论将基于最近开发的计算方法来解释相互作用，并将结合到描述相似和不相似蛋白质之间相互作用的平均力（PMF）的精确势中。 该PMF是描述蛋白质溶液的物理性质和决定相行为的热力学函数的基础。 几种计算和分子模拟方法将被探索用于计算蛋白质溶液的相行为。 我们的工作假设是，蛋白质相互作用的相对大小决定了如何最好地完成特定的分离。一系列实验测量将补充和指导理论。 实验首先涉及使用光散射和小角中子散射（SANS）表征蛋白质的结构和功能特性。 接下来，将进行蛋白质的热力学和物理性质以及混合物的测量，以测试热力学描述的有效性。 特别感兴趣的一个热力学变量将是压力。 具体来说，研究者将研究压力对蛋白质稳定性的影响，以及如何通过压力操纵蛋白质折叠以控制蛋白质分离和纯化。 最后，将进行分离实验，以评估基于热力学模型的性能预测。 这些测量和计算将指导寻找不利相互作用损害分离效果的条件，并设计和测试适当的补救措施。 将检查几种蛋白质和蛋白质混合物，包括eglin C和葡萄球菌核酸酶。这种实验和理论工具的结合将确定在分离过程的设计中重要的蛋白质的性质，并将允许分离性能的蛋白质性质的基础上进行预测。 该项目是特拉华州大学（0078844）研究人员小组提案的一部分。