PEG-WATER SYSTEM, A MODEL FOR CELLULAR ENVIRONMENT

PEG-WATER 系统，细胞环境模型

• 批准号: 3288016
• 负责人: ANDREAS S ROSENBERG
• 金额: $ 6.51万
• 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/3288016
• 关键词: acidity /alkalinity; carbonate dehydratase; conformation; cytoplasm; ethylene glycols; hemoglobin; lysozyme; microcalorimetry; molecular weight; nonradiation isotope effect; radiotracer; solubility; solutions; 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。 热力学活性及其水合状态。 大多数研究表明， 行为已经在溶液条件下进行， 蛋白质存在的细胞内环境。 在细胞质中，蛋白质 浓度高，溶液状态明显不理想。 我们 计划剖析影响这些解决方案条件的因素，特别是 排除体积和水活性，使用蛋白质/PEG/水模型 我们建议密切接近的解决方案的条件， 胞质液 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， 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，当两个血红蛋白，在其脱氧状态， 沉淀在一起，混合物的溶解度显著增加。 高于通过两种物质的组合溶解度所预期的 件. 将使用各种纯化的蛋白质并直接 将进行固相和溶液相的分析。 对于一些 实验中，我们将使用蛋白质通过一个6-碳连接到凝胶颗粒， 间隔物（以避免由于蛋白质聚集而引起的问题）。 技术 蛋白质相互作用的热力学分析， 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 实验，就其性质而言，将需要相对长期的努力 因此，我们计划进行一项为期5年的研究。 我们的方法将首先解决 为以后的研究打下良好的基础。 在 在这一阶段，我们将表征水/PEG的物理性质， 水/PEG/蛋白质溶液的H+和H-活性，水 活性（通过蒸气压测量）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）在PEG存在下蛋白质的稳定性;（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 溶液条件近似的胞质溶胶，使用 水/PEG/蛋白质模型系统。