PEG-WATER SYSTEM, A MODEL FOR CELLULAR ENVIRONMENT

PEG-WATER 系统，细胞环境模型

• 批准号: 3288017
• 负责人: ANDREAS S ROSENBERG
• 金额: $ 6.89万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-07-01 至 1990-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3288017
• 关键词: acidity /alkalinity; carbonate dehydratase; conformation; cytoplasm; ethylene glycols; hemoglobin; lysozyme; microcalorimetry; molecular weight; nonradiation isotope effect; protein sequence; radiotracer; solubility; solutions; spectrin; thermodynamics; tritium; viscosity; water

Protein's behavior, such as enzyme activity, is a function of their thermodynamic activity and their state of hydration. Most studies of their behavior have been conducted in solution conditions far different from the intracellular environment which proteins exist. In the cytosol, protein concentration is high and the solution in state is markedly non-ideal. We plan to dissect the factors influencing these solution conditions, notably excluded volume and water activity, using a model of protein/PEG/water which we suggest closely approximates the solution conditions of the cytosol. Our aim then is to study how these conditions influence protein-protein interaction, protein-ligand interaction and the conformational stability and motility of proteins. In preliminary studies, we have found that when two hemoglobins, in their deoxy states, are precipitated together, the solubility of the mixture is significantly higher than would be expected by the combined solubilities of the two components. A variety of purified proteins will be used and direct analysis of the solid and solution phases will be made. For some of the experiments we will use proteins tethered to gel particles by a 6-carbon spacer (in order to avoid problems due to protein aggregation). Techniques of analysis of the thermodynamics of protein-protein interaction and of conformational mobility which we will use will include microcalorimetric analysis, hydrogen exchange and the measurement of ligand-binding. Our experiments, by their nature, will require a relatively long term effort and thus we are projecting a 5 year study. Our approach will first address itself to the formation of a satisfactory base for subsequent studies. In this phase, we will characterize the physical properties of water/PEG and water/PEG/protein solutions with respect to H+ and H- activities, water activity (by vapor pressure measurements) and surface tension. In the second phase, which will be more in line with our ultimate aims, we will study: (a) the stability of proteins in the presence of PEG; (b) the formation of mixed solid states, including monomer-polymer assembly; and (c) protein-ligand interactions. All of these studies will be done under solution conditions approximating those of the cytosol, using the water/PEG/protein model system.

蛋白质的行为，如酶的活性，是它们的功能 热力学活性和它们的水合状态。大多数关于他们的研究 行为是在溶液条件下进行的，与 蛋白质存在的细胞内环境。在胞浆中，蛋白质 浓度高，溶液状态明显不理想。我们 计划分析影响这些解决方案条件的因素，尤其是 排除体积和水活度，使用蛋白质/聚乙二醇水模型 我们建议它非常接近于 胞浆。我们的目标是研究这些条件如何影响 蛋白质-蛋白质相互作用、蛋白质-配体相互作用和 蛋白质的构象稳定性和运动性。在初步研究中， 我们已经发现，当两种脱氧态的血红蛋白 沉淀在一起，混合物的溶解度显著 比两者的溶解度之和预期的要高 组件。将使用和直接使用各种纯化的蛋白质 将对固体相和溶液相进行分析。对于某些人来说 实验中，我们将使用蛋白质通过6-碳来凝胶化颗粒 间隔物(以避免蛋白质聚集带来的问题)。技术 蛋白质相互作用的热力学分析和蛋白质相互作用的热力学 我们将使用的构象迁移率将包括微量热 分析、氢交换和配体结合的测量。我们的 从本质上讲，实验需要相对长期的努力。 因此，我们计划进行为期5年的研究。我们的方法将首先解决 为后续研究形成了一个令人满意的基础。在……里面 在这一阶段，我们将表征水/聚乙二醇的物理性质和 水/聚乙二醇/蛋白质溶液对H+和H-活度的影响 活度(通过蒸汽压测量)和表面张力。在 第二阶段，这将更符合我们的最终目标，我们将 研究：(A)蛋白质在聚乙二醇存在下的稳定性；(B) 形成混合固态，包括单体-聚合物组装；以及 (C)蛋白质-配体相互作用。所有这些研究都将在 溶液条件近似于胞液的溶液条件，使用 水/聚乙二醇/蛋白质模型体系。