THERMODYNAMIC AND NMR STUDIES ON THE E COLI CYTOPLASM

大肠杆菌细胞质的热力学和核磁共振研究

• 批准号: 2184504
• 负责人: M. THOMAS RECORD
• 金额: $ 17.37万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-01 至 1996-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2184504
• 关键词: DNA binding protein; Escherichia coli; betaine compound; biophysics; cell growth regulation; cell water; chemical kinetics; computer simulation; cytoplasm; electrolyte balance; growth media; high performance liquid chromatography; intermolecular interaction; light scattering; mathematical model; nuclear magnetic resonance spectroscopy; nucleic acids; osmotic pressure; polyion; potassium; proline; putrescine; solute; spermidine; temperature sensitive mutant; thermodynamics; tissue /cell culture; trehalose

The long-term goal of our research is to understand the thermodynamic basis of cytoplasmic function. In particular, we focus on the massive intracellular nonideality due to polyelectrolyte effects (cation accumulation at the surface of nucleic acids), preferential interactions (accumulation/exclusion) of solutes with proteins, excluded volume effects (crowding) and solvent nonideality (macromolecular hydration). We seek to determine the key physical chemical properties that govern the equilibria and kinetics of noncovalent interactions of proteins and of nucleic acids in the cytoplasm. This research addresses directly the important problem of relating in vitro studies of the thermodynamics and kinetics of processes of biological molecules to their in vivo function. Only after the composition and relevant thermodynamic properties of the intracellular environment have been quantified will it be possible to simulate these properties reliably in vitro or to extrapolate correctly from measurements made in a dilute solution in vitro to the much more concentrated in vivo state. Motivated by the long-term goals summarized above, our specific aims encompass three tightly-interrelated areas: 1) obtaining the necessary data; 2) developing the relevant statistical thermodynamic framework in order to analyze these data and evaluate thermodynamic coefficients, and 3) using these thermodynamic coefficients to predict the consequences of extreme thermodynamic nonideality for cellular processes as a function of osmolarity. In particular, we are investigating the thermodynamic bases of a) an in vivo-in vitro paradox (first noted by us) regarding the absence of [salt]-effects on gene expression in vivo, b) the quantitative linkage (also first noted by us) between effects of osmolarity and osmoprotectants on growth rate, cytoplasmic K+ activity and cytoplasmic water volume, and c) the behavior of "osmotic remedial" mutants, which constitute a large sub-class of conditional-lethal mutants in which the wild-type phenotype is restored by growth at high osmolarity. Our novel thermodynamic proposals to explain these and other aspects of cell function represent the first of what we expect to be numerous advances in the application of classical and statistical thermodynamics to the analysis of in vivo processes. Key physiological variables which we utilize to change the thermodynamic state of the cytoplasm are: a) external osmolarity and b) the presence or absence of "osmoprotectants" (e.g. glycine betaine, proline) in the growth medium. To obtain thermodynamic information for cells and for cytoplasmic species, we will measure: a) volumes of cytoplasmic water in unplasmolyzed cells and in cells titrated with a plasmolyzing agent (e.g. NaCl); b) osmotic and turgor pressures, and osmotic coefficients of the cytoplasm; c) amounts, osmotic properties, and NMR characteristics of osmotically- regulated cytoplasmic species; d) amounts of electrolyte ions, oligocations and polyanions responsible for cytoplasmic electroneutrality; and e) amounts and states of association of cytoplasmic macromolecules responsible for crowding.

我们研究的长期目标是了解热力学基础 细胞质功能。 特别是，我们专注于大规模 由于聚电解质效应引起的细胞内非理想性（阳离子 在核酸表面积聚），优先相互作用 （累积/排除）蛋白质的溶质，排除体积效应 （拥挤）和溶剂非理想性（大分子水合）。 我们寻求 确定控制平衡的关键物理化学特性 蛋白质和核酸的非共价相互作用的动力学 在细胞质中。 这项研究直接解决了重要的问题 关于热力学和动力学的体外研究 生物分子的过程到其体内功能。 只有之后 细胞内的组成和相关热力学特性 环境已被量化是否可以模拟这些 在体外可靠或从测量中正确推断特性 在体外稀释的稀释溶液中制成 状态。 我们的具体目的是受上述长期目标的激励 包含三个紧密间隔的区域：1）获得必要的 数据; 2）开发相关的统计热力学框架 为了分析这些数据并评估热力学系数，3） 使用这些热力学系数预测 针对细胞过程的极端热力学非理想性 渗透性。 特别是，我们正在研究 a）关于不存在的体内体外体外悖论（我们首先指出） [盐]对体内基因表达的影响，b）定量键 （也首先是我们指出的）在渗透性和渗透保护剂的效果之间 关于生长速率，细胞质K+活性和细胞质水体积，以及 c）“渗透补救”突变体的行为，构成一个大的 野生型表型的有条件致命突变体的子类 在高渗透压下通过生长恢复。 我们的新型热力学建议 解释这些细胞功能的这些和其他方面代表 我们期望在经典和 统计热力学分析体内过程。 我们用来改变热力学的关键生理变量 细胞质的状态为：a）外渗透压和b）存在或 在生长中没有“渗透保护剂”（例如甘氨酸，脯氨酸） 中等的。 获得细胞和细胞质的热力学信息 物种，我们将测量：a）垂液中的细胞质水量 细胞和用浆液剂（例如NaCl）滴定的细胞； b） 渗透压和杂物的压力以及细胞质的渗透系数； c） 渗透的量，渗透特性和NMR特性 调节的细胞质物种； d）电解质离子的量 以及负责细胞质电压的多镜；和e） 负责的细胞质大分子的数量和状态 拥挤。