Solute Effects on Biopolymer Processes

描述(申请人提供)：在体外，试错法用于选择溶质来优化或扰乱生物聚合物过程(例如折叠、组装、结晶、结合)。我们的长期目标是开发定量方法，根据埋藏或暴露的生物聚合物表面的数量和类型来预测或解释溶质对生物聚合物过程的影响。为此，正在开发蒸汽压渗透仪来测量变性剂、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表面和极性表面的相互作用。这些溶质与相对不带电荷、极性较低的生物聚合物表面的相互作用是通过它们对选定的生物聚合物构象变化(边缘稳定的多肽和蛋白质的展开；核酸螺旋的融化)的影响来量化的。从这些数据中获得的有关溶质与带电、极性和非极性生物聚合物表面相互作用的定量信息将被用来从结构上预测或解释溶质效应。对尿素和甘氨酸甜菜碱的计算结果表明，对于两种截然不同的溶质，这种定量方法具有优势。在体内，细胞质中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的水分、体积和渗透压。我们观察到胞质水的量以及生物聚合物和溶质的胞质浓度(例如：[K+]，[甘氨酸甜菜碱])。对于所考察的所有条件，这些变化与生长速度的变化相关。为了测试这些相关性并探索它们的分子基础，在恒定渗透压下，使用不同的渗透保护剂和使用缺少一种渗透保护剂转运蛋白的菌株，细胞质溶质组成将发生变化。将确定对生长速度和细胞质水分和溶质数量的影响，并将其用于体积调节和补偿机制的定量模型，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+].