High-throughput assay for protein-ligand interaction

描述（由申请人提供）：蛋白质与低分子量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检测，难以适应高通量筛选，以鉴定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毛细管作用差异的新型测定方法。初步研究表明，差分径向毛细管试验可以检测细菌次级信号分子环二gmp （cdiGMP）和Alg44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检测特异性。此外，差分径向毛细管分析允许定量测量cdiGMP与Alg44的解离常数和解离速率，这与先前发表的报告相匹配。此外，该实验还可以检测cdiGMP与表达Alg44受体的细胞的全细胞裂解物的相互作用。本提案的目的是探讨差异径向毛细管测定法的局限性，以及该测定法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，我们将确定差分径向毛细管检测其他蛋白质-配体对的特异性和准确性，兼容配体的范围，检测生化反应的能力以及异种表达蛋白质的溶解度要求。在目标2中，我们将研究该方法在全细胞系统中的检测极限，并确定其他模型蛋白表达系统（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，我们将通过系统筛选铜绿假单胞菌和霍乱弧菌orfeome，应用该方法鉴定新的cdiGMP结合蛋白。这些研究结果将对理解cdiGMP的调控和发展功能代谢组学领域具有广泛的意义。

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.