High-throughput assay for protein-ligand interaction

蛋白质-配体相互作用的高通量测定

• 批准号: 8176077
• 负责人: VINCENT T LEE
• 金额: $ 21.15万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-15 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8176077
• 关键词: Abscisic Acid; Agriculture; Allosteric Regulation; Animals; Auxins; Bacteria; Binding; Binding Proteins; Biochemical; Biochemical Reaction; Biological; Biological Assay; Biology; Blood capillaries; Bypass; Calcium; Calorimetry; Cataloging; Catalogs; Cell Extracts; Cells; Cyclic AMP; Cyclic GMP; Cyclic Nucleotides; Cytolysis; Data; Detection; Development; Disease; Dissociation; Drug Design; Drug usage; Endocrine system; Equipment; Escherichia coli; Goals; Hormones; Imagery; Inclusion Bodies; Individual; Insecta; Intervention; Ligands; Mammalian Cell; Measurement; Measures; Membrane; Methods; Modeling; Molecular Weight; Neurotransmitters; Pharmaceutical Preparations; Pharmacologic Substance; Plants; Procedures; Process; Protein Analysis; Proteins; Pseudomonas aeruginosa; Publishing; Pyroxylin; Radial; Regulation; Reporting; Saccharomyces; Saccharomyces cerevisiae; Sampling; Screening procedure; Signal Pathway; Signal Transduction; Signaling Molecule; Solubility; Specificity; Surface Plasmon Resonance; System; Technology; Testing; Time; Vibrio cholerae; Work; base; biological systems; capillary; high throughput analysis; high throughput screening; inhibitor/antagonist; interest; metabolomics; novel; protein S precursor; protein expression; rapid detection; receptor; research study; small molecule

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.

描述（由申请人提供）：与低分子量配体的蛋白质相互作用对生物学对变构调节和酶促活性都有很大的影响。蛋白质 - 配体相互作用的另一个重要方面是针对药物的开发及其对生物系统的预期和非特异性影响。但是，当前的技术仅限于难以适应高通量筛选的特定测定法，以识别新型蛋白质受体和小分子抑制剂。为了绕过这些局限性，我们基于差异径向毛细管作用开发了一种新颖的测定法。最初的工作表明，差异径向毛细管测定可以检测细菌二级信号分子，环状-DI-GMP（CDIGMP）和ALG44受体蛋白之间的相互作用。该测定允许基于未标记配体的竞争实验检测特异性。此外，差异径向毛细管测定法可以定量测量CDIGMP与ALG44的分离常数和解离速率，该速率与先前发表的报告相匹配。此外，该测定可以检测到表达ALG44受体的细胞中CDIGMP与全细胞裂解物的相互作用。该提案的目标是探讨差异径向毛细管测定的局限性以及该测定法对蛋白质配体相互作用的高通量筛选的适用性。在AIM 1中，我们将确定针对其他蛋白质配对的差异径向毛细管测定的特异性和准确性，兼容配体的范围，检测生化反应的能力以及异源表达的蛋白质的溶解度需求。在AIM 2中，我们将研究整个细胞系统中测定的检测极限，并确定其他模型蛋白表达系统（例如酿酒酵母，昆虫细胞和哺乳动物细胞）是否与测定兼容。此外，我们将通过系统地筛选铜绿假单胞菌和弧菌霍乱甲状腺虫的拟南芥结合蛋白来鉴定新颖的CDIGMP结合蛋白。这些研究的结果将对理解CDIGMP调节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：鉴定蛋白质 - 配体相互作用的能力对于了解生物系统的调节以及药物治疗至关重要。该提案旨在开发高通量差异径向毛细管测定法，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与药物与蛋白质受体之间的功能相互作用提供基础。