Solute Effects on Biopolymer Processes

描述（由申请人提供）：在体外，试错法用于选择溶质来优化或干扰生物聚合物过程（例如折叠、组装、结晶、结合）。我们的长期目标是发展定量方法来预测或解释溶质对生物聚合物过程的影响，根据埋藏或暴露的生物聚合物表面的数量和类型。为此，蒸汽压渗透法正在发展，以测量变性剂、渗透剂、结晶剂和霍夫迈斯特盐与天然蛋白质和核酸的相对带电和极性表面的相互作用。这些溶质与相对不带电、极性较低的生物聚合物表面的相互作用，通过它们对选定的生物聚合物构象变化（边缘稳定的肽和蛋白质的展开；核酸螺旋的融化）的影响来量化。从这些数据中获得的溶质与带电、极性和非极性生物聚合物表面相互作用的定量信息将用于预测或解释结构方面的溶质效应。尿素和甘氨酸甜菜碱的结果显示了这种定量方法对两种非常不同的溶质的优点。在体内，细胞质溶质的数量及其与生物聚合物和溶质的相互作用完全决定了细胞质水的数量、体积和渗透压。我们观察到，随着渗透渗透压的变化或渗透保护剂的加入，大肠杆菌中细胞质水的数量以及生物聚合物和溶质（如[K+]、[甜菜碱]）的浓度发生了很大的变化。在所有条件下，这些变化都与生长速率的变化相关。为了测试这些相关性并探测其分子基础，将使用不同的渗透保护剂和缺乏一种渗透保护剂的菌株，在恒定的渗透压下改变细胞质溶质组成。将确定对生长速率和细胞质水和溶质数量的影响，并将其用于体积调节和补偿机制的定量模型中，以减少细胞质[K+]变化的扰动效应。

DESCRIPTION (provided by applicant): In vitro, trial-and-error is used to select solutes to optimize or perturb biopolymer processes (e.g. folding, assembly, crystallization, binding). Our long term goal is to develop quantitative methods to predict or interpret solute effects on biopolymer processes in terms of the amounts and types of biopolymer surfaces buried or exposed. To this end, vapor pressure osmometry is being developed to measure interactions of denaturants, osmolytes, crystallization agents and Hofmeister salts with the relatively charged and polar surfaces of native proteins and nucleic acids. Interactions of these solutes with relatively uncharged, less polar biopolymer surfaces are quantified from their effects on selected biopolymer conformational changes (unfolding of marginally stable peptides and proteins; melting of nucleic acid helices). The quantitative information about interactions of solutes with types of charged, polar and nonpolar biopolymer surface obtained from this data will be used to predict or interpret solute effects in terms of structure. Results for urea and glycine betaine show the merit of this quantitative approach for two very different solutes. In vivo, amounts of cytoplasmic solutes and their interactions with biopolymers and solutes completely determine the amount of cytoplasmic water, volume, and osmolality. We observe large changes in the amount of cytoplasmic water and in cytoplasmic concentrations of biopolymers and solutes (e.g. [K+], [glycine betaine]) in E. coli upon shifts in osmolality or addition of osmoprotectants. For all conditions examined, these changes correlate with changes in growth rate. To test these correlations and to probe their molecular basis, cytoplasmic solute composition will be varied at constant osmolality using different osmoprotectants and using strains lacking one osmoprotectant porter. Effects on growth rate and on amounts of cytoplasmic water and solutes will be determined, and used in quantitative models of volume regulation and of compensation mechanisms to reduce the perturbing effects of changes in cytoplasmic [K+].