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，我们关注的是大规模的 聚电解质效应(阳离子)引起的细胞内非理想性 在核酸表面的积累)，优先相互作用 (累积/排除)含有蛋白质的溶质，排除体积效应 (拥挤)和溶剂非理想性(大分子水合)。我们寻求 确定支配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这一重要问题。 相关的热力学和动力学的体外研究 生物分子的过程对其体内功能的影响。仅在之后 细胞内的组成和相关的热力学性质 环境已经被量化了，是否有可能模拟这些 在体外可靠地或从测量中正确推断的性质 在体外制成的稀溶液在体内要浓缩得多 州政府。 在上述长期目标的激励下，我们的具体目标 包括三个紧密相关的领域：1)获得必要的 数据；2)开发相关的统计热力学框架 以分析这些数据并评估热力学系数，以及3) 使用这些热力学系数来预测 作为函数的细胞过程的极端热力学非理想性 渗透压。尤其是，我们正在研究的热力学基础 A)体内-体外悖论(由我们最先注意到)，关于缺乏 [盐]-对体内基因表达的影响，b)数量连锁 (也是我们最先注意到的)渗透压和渗透保护剂的影响 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 条件致死突变体的亚类，其中野生型表型是 通过高渗透压下的生长恢复的。我们的新热力学建议 为了解释这些和细胞功能的其他方面，代表了第一个 我们所期待的是在古典音乐和音乐的应用方面取得的巨大进步 统计热力学用于活体过程的分析。 我们用来改变热力学的关键生理变量 细胞质的状态是：a)外渗透压和b)存在或 生长过程中缺乏“渗透保护剂”(如甘氨酸甜菜碱、脯氨酸) 5~6成熟。获取细胞和细胞质的热力学信息 物种，我们将测量：a)未质化的细胞质水的体积 细胞和用胞浆增效剂(例如，氯化钠)滴定的细胞；b) 渗透压、膨压和细胞质渗透系数；c) 渗透剂的量、渗透性能和核磁共振特性 受调控的细胞质种类；d)电解质离子的数量、寡聚位置 和负责细胞质电中性的多阴离子；和e) 相关细胞质大分子的缔合量和结合状态 用来挤人。

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.