DEVELOPING SOLUTES AS STRUCTURAL/MECHANISTIC PROBES OF PROTEIN-DNA INTERACTIONS

开发溶液作为蛋白质-DNA 相互作用的结构/机械探针

• 批准号: 8389868
• 负责人: M. THOMAS RECORD
• 金额: $ 28.77万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8389868
• 关键词: Agreement; Alanine; Amides; Ammonium Sulfate; Anions; Area; Betaine; Binding; Biochemical; Biochemistry; Biological Process; Biomedical Engineering; Biophysics; Biopolymers; Burial; Carbon; Cations; Cell Volumes; Cells; Charge; Chemicals; Circular Dichroism; Communities; Coupled; Crystallization; DNA; DNA Binding Domain; DNA Probes; DNA Sequence; DNA-Binding Proteins; DNA-Protein Interaction; Data; Databases; Dehydration; Development; Disease; Escherichia coli; Ethylene Glycols; Goals; Growth; Hydration status; Hydrogen Bonding; Ions; Kinetics; Lac Repressors; Measurement; Measures; Microscopic; Modeling; Molecular; Nitrogen; Nucleic Acids; Oxygen; Partition Coefficient; Peptides; Pharmacologic Substance; Polyethylene Glycols; Process; Proline; Property; Proteins; Publishing; Regulation; Relative (related person); Research; Role; Salts; Sodium Chloride; Solubility; Spectrum Analysis; Stress; Surface; Testing; Thermodynamics; Time; Trehalose; Trifluoroethanol; Urea; Water; base; ethylene glycol; functional group; in vivo; inorganic phosphate; insight; interest; melting; novel; physical chemical interaction; protein aggregation; protein misfolding; public health relevance; research study; response; self assembly; small molecule; solute; success

DESCRIPTION (provided by applicant): Biochemically-significant small molecules (Hofmeister salts, osmolytes, denaturants, collectively referred to as solutes) exert often large, specific effects on fundamental biological processes involving proteins and nucleic acids (e.g. folding, assembly, binding, aggregation). The long term goal of this project is to interpret and predict these effects quantitatively in terms of structural information, and to develop these solutes as probes of interface formation and coupled folding in processes lacking structural information. Insight into the roles of E. coli osmolytes (e.g. Kglutamate, glycine betaine) in the biophysics of regulation of cytoplasmic volume and biopolymer volume fraction in response to osmotic stress will be obtained. Roles of osmolytes in diseases caused by protein misfolding and aggregation in vivo have recently been proposed; these are just one manifestation of what must be widespread, significant effects of changes in type and concentration of intracellular solutes on cellular biochemistry. To achieve these goals, we propose to obtain and interpret quantitative thermodynamic data characterizing interactions of solutes with biopolymer and model surfaces and effects of these solutes on processes. Data will be analyzed using the recently-developed solute partitioning model (SPM), which interprets effects of a solute on a biopolymer process in terms of the change in water accessible surface area of the biopolymer and the local excess or deficit in concentration of the solute in the water of hydration of that surface. The latter is quantified by a microscopic partition coefficient Kp, a property of both the solute and the composition (%nonpolar, polar, charged) of the surface. By determining overall Kp for processes exposing or burying surfaces with widely different compositions, we will build up a database of partition coefficients Kp to predict solute or salt effects on processes where structural data is available. Experiments are proposed to characterize interactions of solutes with uncharged biopolymer surfaces exposed upon unfolding/melting, including circular dichroism studies with marginally-stable proteins (alpha-helical alanine-based peptides and the globular DNA-binding-domain of lac repressor) and UV-melting studies with a marginally stable 12 bp DNA duplex. To interpret these data, osmometric or solubility studies of interactions of these solutes with appropriately chosen model compounds will be performed. In the longer term, Hofmeister salt and solute effects on significant processes burying charged as well as uncharged biopolymer surface (e.g. protein crystallization, protein-nucleic acid interactions) will also be studied. This research will allow effects of Hofmeister salts and solutes, including denaturants GuHCl and urea, osmolytes Kglutamate and glycine betaine, crystallization agents ammonium sulfate, MPD and PEG, and helix stabilizers like TFE, on any protein, nucleic acid or model process to be quantitatively predicted for the first time in terms of structural information.

描述（由申请人提供）：具有生物化学意义的小分子（霍夫迈斯特盐、渗透剂、变性剂，统称为溶质）通常对涉及蛋白质和核酸的基本生物过程（例如折叠、组装、结合、聚集）产生较大的特异性影响。该项目的长期目标是根据结构信息定量解释和预测这些效应，并开发这些溶质作为缺乏结构信息的过程中界面形成和耦合折叠的探针。对E.将获得大肠杆菌渗透调节物（例如谷氨酸钾、甘氨酸甜菜碱）在响应渗透胁迫调节细胞质体积和生物聚合物体积分数的生物物理学中的作用。渗透调节剂在体内蛋白质错误折叠和聚集引起的疾病中的作用最近被提出;这些只是细胞内溶质的类型和浓度变化对细胞生物化学的广泛、显著影响的一种表现。 为了实现这些目标，我们建议获得和解释定量的热力学数据，表征溶质与生物聚合物和模型表面的相互作用，以及这些溶质对过程的影响。数据将使用最近开发的溶质分配模型（SPM）进行分析，该模型解释了溶质对生物聚合物过程的影响，即生物聚合物的水可及表面积的变化以及该表面的水合水中溶质浓度的局部过量或不足。后者通过微观分配系数Kp定量，这是溶质和表面组成（%非极性，极性，带电）的性质。通过确定暴露或掩埋具有广泛不同组成的表面的过程的总体Kp，我们将建立分配系数Kp的数据库，以预测溶质或盐对结构数据可用的过程的影响。实验提出了表征的相互作用的溶质与不带电的生物聚合物表面暴露后展开/熔化，包括圆二色性研究与边缘稳定的蛋白质（α-螺旋丙氨酸为基础的肽和球状DNA结合域的乳糖阻遏物）和UV熔化的研究与边缘稳定的12 bp的DNA双链体。为了解释这些数据，将对这些溶质与适当选择的模型化合物的相互作用进行渗透压或溶解度研究。从长远来看，霍夫迈斯特盐和溶质对掩埋带电和不带电生物聚合物表面（例如蛋白质结晶，蛋白质-核酸相互作用）的重要过程的影响也将进行研究。这项研究将允许霍夫迈斯特盐和溶质，包括变性剂盐酸胍和尿素，渗透剂谷氨酸钾和甘氨酸甜菜碱，结晶剂硫酸铵，MPD和PEG，以及螺旋稳定剂如TFE，对任何蛋白质，核酸或模型过程的影响首次在结构信息方面进行定量预测。