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

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、渗透变性剂、变性剂，统称为溶质)对涉及蛋白质和核酸的基本生物过程(如折叠、组装、结合、聚集)往往产生巨大的、特定的影响。该项目的长期目标是根据结构信息定量解释和预测这些影响，并将这些溶质开发为缺乏结构信息的过程中界面形成和耦合折叠的探针。深入了解大肠杆菌渗透调节剂(如谷氨酸、甘氨酸甜菜碱)在调节细胞质体积和生物聚合物体积分数以响应渗透胁迫的生物物理中的作用。渗透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类型和浓度变化对细胞生物化学的广泛、显著影响的一个表现。为了实现这些目标，我们建议获得并解释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的定量热力学数据。数据将使用最近开发的溶质分配模型(SPM)进行分析，该模型根据生物聚合物的水可达表面积的变化以及该表面水化水中溶质浓度的局部过剩或不足来解释溶质对生物聚合物过程的影响。后者由微观分配系数Kp来量化，Kp是溶质和表面成分(非极性、极性、带电的百分比)的属性。通过确定暴露或埋藏成分差异很大的表面的过程的总体Kp，我们将建立一个分配系数Kp的数据库，以预测溶质或盐对可获得结构数据的过程的影响。建议用实验来表征溶质与未带电的生物聚合物表面在展开/熔融时的相互作用，包括与边缘稳定的蛋白质(基于α-螺旋丙氨酸的多肽和lac阻遏物的球形DNA结合域)的圆二色研究，以及与边缘稳定的12个碱基DNA双链的紫外熔融研究。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渗透或溶解度研究。从长远来看，霍夫迈斯特盐和溶质对埋藏带电和不带电生物聚合物表面的重要过程(例如蛋白质结晶、蛋白质-核酸相互作用)的影响也将被研究。这项研究将使Hofmeister盐和溶质，包括变性剂盐酸古胺和尿素，渗透剂谷氨酸和甘氨酸甜菜碱，结晶剂硫酸铵，MPD和聚乙二醇酯，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从结构信息方面进行定量预测。 与公共健康相关：这项研究开发了一种新颖的一般策略，以测量、解释并最终预测Hofmeister盐对蛋白质和核酸过程(例如折叠、自组装、结合、结晶)的小溶质(例如变性剂、渗透压)和非库仑效应的影响，方法是根据这些过程中埋藏或暴露在水中的生物聚合物表面的数量和组成的结构信息。通过取代“试验和错误”，这项研究产生的数据库将促进使用溶质和霍夫迈斯特盐来优化或探索蛋白质和核酸过程的步骤，并将有助于开发溶质(例如作为稳定剂)的制药或生物工程应用。此外，这些结果将有助于我们了解细胞内溶质之间的相互作用以及与生物聚合物的相互作用，生物聚合物是细胞内水分、细胞体积、生物聚合物体积分数的关键决定因素，甚至在给定的生长条件下的生长速度。

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. PUBLIC HEALTH RELEVANCE: This research develops a novel and general strategy to measure, interpret and eventually predict effects of small solutes (e.g. denaturants, osmolytes) and noncoulombic effects of Hofmeister salts on protein and nucleic acid processes (e.g. folding, self-assembly, binding, crystallization) in terms of structural information on the amount and composition of the biopolymer surface buried or exposed to water in these processes. By replacing "trial and error", the database generated by this research will facilitate the use of solutes and Hofmeister salts to optimize or probe the steps of protein and nucleic acid processes, and will aid in the development of pharmaceutical or bioengineering applications of solutes (e.g. as stabilizers). In addition, these results will help us understand the interactions of intracellular solutes with each other and with biopolymers which are a key determinant of amount of water in the cell, cell volume, biopolymer volume fraction and even growth rate at a given growth condition.