THERMODYNAMIC AND NMR STUDIES ON THE E COLI CYTOPLASM

我们研究的长期目标是了解热力学基础 细胞质的功能。 特别是，我们专注于大规模的 细胞内非理想性，由于质子效应（阳离子 在核酸表面的积累），优先相互作用 溶质与蛋白质的（蓄积/排除），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性质 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一个重要的问题， 将体外研究的热力学和动力学 生物分子在体内的功能。 后才 细胞内的组成和相关的热力学性质 环境已经量化，将有可能模拟这些 可靠地在体外或从测量中正确地外推性能 从体外的稀释溶液到体内的浓缩溶液 状态 基于上述长期目标，我们的具体目标 包括三个密切相关的领域：1）获得必要的 数据; 2）发展相关的统计热力学框架， 以便分析这些数据并评估热力学系数，以及3） 用这些热力学系数来预测 细胞过程的极端热力学非理想性， 渗透压 特别是，我们正在研究的热力学基础， a）关于缺乏的体内-体外悖论（我们首先注意到） [盐]-对体内基因表达的影响，B）定量关联 (also我们首先注意到）渗透压和细胞保护剂之间的影响 对生长速率、细胞质K+活性和细胞质水体积的影响， c）“渗透补救”突变体的行为，这构成了一个大的 条件致死突变体的子类，其中野生型表型是 通过在高渗透压下生长而恢复。 我们新的热力学建议 为了解释细胞功能的这些和其他方面， 我们期待的是在经典和 统计热力学用于分析体内过程。 我们用来改变热力学的关键生理变量 细胞质的状态是：a）外部渗透压和B）存在或 在生长中不存在“植物保护剂”（例如甘氨酸甜菜碱、脯氨酸） 介质 获得细胞和细胞质的热力学信息 物种，我们将测量：a）体积的细胞质水在未塑化 细胞和用增塑剂（例如NaCl）滴定的细胞中; B） 渗透压和膨压，以及细胞质的渗透系数; c） 量，渗透性能，和NMR特征， 调节的细胞质种类; d）电解质离子、寡阳离子的量 和负责细胞质电中性的聚阴离子;和 细胞质大分子的数量和状态 因为拥挤。

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.