Solute Effects on Biopolymer Processes

溶质对生物聚合物过程的影响

• 批准号: 7928500
• 负责人: M. THOMAS RECORD
• 金额: $ 10.65万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7928500
• 关键词: Alanine; Amides; Area; Betaine; Binding; Biological Assay; Biopolymers; Cell physiology; Charge; Crystallization; Culture Media; Cytoplasm; Data; Development; Escherichia coli; Exclusion; Financial compensation; Gene Expression; Glycine; Goals; Growth; In Vitro; Investigation; Measures; Methods; Modeling; Molecular; Nucleic Acids; Nucleoproteins; Osmolalities; Oxygen; Peptides; Physiological; Process; Proteins; Regulation; Research Personnel; Salts; Stress; Structure; Surface; Testing; Thermodynamics; Time; Urea; Water; base; globular protein; in vivo; melting; mutant; novel; novel strategies; pressure; programs; radiochemical; solute; vapor

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+].

描述（由申请人提供）：在体外，通过反复试验来选择溶质以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，正在开发蒸气压渗透压测定法来测量变性剂、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和极性表面的相互作用。这些溶质与相对不带电、极性较小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；核酸螺旋的解链）的影响来量化。从该数据获得的关于溶质与带电、极性和非极性生物聚合物表面类型的相互作用的定量信息将用于预测或解释溶质在结构方面的影响。尿素和甘氨酸甜菜碱的结果显示了这种定量方法对于两种截然不同的溶质的优点。在体内，细胞质溶质的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量、体积和渗透压。我们观察到，当渗透压发生变化或添加渗透保护剂时，大肠杆菌中细胞质水量以及生物聚合物和溶质（例如[K+]、[甘氨酸甜菜碱]）的细胞质浓度发生巨大变化。对于所有检查的条件，这些变化与增长率的变化相关。为了测试这些相关性并探究它们的分子基础，将使用不同的渗透保护剂和使用缺乏一种渗透保护剂转运蛋白的菌株在恒定渗透压下改变细胞质溶质组成。将确定对生长速率以及细胞质水和溶质量的影响，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+] 变化的扰动影响。