High-throughput assay for protein-ligand interaction

描述(由申请人提供)：蛋白质与低分子配体的相互作用在变构调节和酶活性方面具有重要的生物学意义。蛋白质-配体相互作用的另一个重要方面是药物的开发及其对生物系统的预期和非特异性作用。然而，目前的技术仅限于特定的分析，难以适应高通量筛选，从而能够识别新的蛋白质受体和小分子抑制剂。为了绕过这些限制，我们开发了一种基于径向差动毛细管作用的新分析方法。初步工作表明，差示径向毛细管分析可以检测细菌二级信号分子cdiGMP(CdiGMP)与Al44受体蛋白之间的相互作用。该分析允许基于与未标记配体的竞争实验来检测特异性。此外，差示径向毛细管分析可以定量测量cdiGMP与阿尔法44的解离常数和解离速率，这与以前发表的报告一致。此外，该方法还可以检测到cdiGMP与表达阿尔法44受体的细胞的全细胞裂解产物的相互作用。这项建议的目的是探索差示径向毛细管分析的局限性以及该分析在高通量筛选蛋白质-配体相互作用方面的适用性。在目标1中，我们将确定用于其他蛋白质-配基对的差示径向毛细管分析的特异性和准确性、相容配基的范围、检测生化反应的能力以及异源表达蛋白质的溶解性要求。在目标2中，我们将调查该方法在整个细胞系统中的检测极限，并确定其他模式蛋白表达系统，如酿酒酵母、昆虫细胞和哺乳动物细胞是否与该方法兼容。此外，我们将应用该方法通过系统地筛选铜绿假单胞菌和霍乱弧菌ORFeome来鉴定新的cdiGMP结合蛋白。这些研究结果将对了解cdiGMP的调控和发展功能代谢组学领域具有广泛的意义。 公共卫生相关性：识别蛋白质-配体相互作用的能力是理解生物系统调控和药物治疗的核心。这项建议旨在开发一种高通量差示径向毛细管分析，可用于分析这些相互作用。这项工作的结果将为确定代谢物和药物与其蛋白质受体之间的功能相互作用提供基础。

DESCRIPTION (provided by applicant): Protein interactions with low molecular weight ligand have great implication in biology for both allosteric regulation and enzymatic activity. Another important aspect of protein-ligand interaction is for the development of pharmaceuticals and their intended and non-specific effects on biological systems. However, current technology has been limited to specific assays that are difficult to adapt to high-throughput screening to allow identification of novel protein receptors and small molecule inhibitors. To bypass these limitations, we have developed a novel assay based on differential radial capillary action. Initial work has demonstrated the differential radial capillary assay can detect the interaction between a bacterial secondary signaling molecule, cyclic-di-GMP (cdiGMP) and the Alg44 receptor protein. The assay allows detection of specificity based on competition experiments with unlabeled ligands. In addition, the differential radial capillary assay allows for quantitative measurements of dissociation constant and dissociation rate of cdiGMP with Alg44 which matches previously published reports. Furthermore, the assay can detect the interaction of cdiGMP with whole cell lysates from cells expressing the Alg44 receptor. The goals of this proposal are to explore the limitations of the differential radial capillary assay and the applicability of the assay for high-throughput screening for protein-ligand interactions. In Aim 1, we will determine the specificity and accuracy of the differential radial capillary assay for other protein-ligand pairs, the range of compatible ligands, the ability to detect biochemical reactions and the solubility requirement for heterologously expressed proteins. In Aim 2, we will investigate the limit of detection of the assay in whole cell system and determine if other model protein expressing systems, such as Saccharomyces cereviciae, insect cells and mammalian cells, are compatible with the assay. Furthermore, we will apply the assay to identify novel cdiGMP binding proteins by systematically screening the Pseudomonas aeruginosa and Vibrio cholerae ORFeomes. The results from these studies will have broad implication for the understanding of cdiGMP regulation and developing the field of functional metabolomics. PUBLIC HEALTH RELEVANCE: The ability to identify protein-ligand interactions is central to understanding regulation of biological systems as well as pharmaceutical treatment. This proposal seeks to develop a high-throughput differential radial capillary assay that can be used to analyze these interactions. Results from this work will provide a basis for determining the functional interactions between metabolites and drugs with their protein receptors.