DEVELOPMENT AND APPLICATION OF PLIMSTEX

PLIMSTEX的开发与应用

• 批准号: 7721419
• 负责人: MICHAEL L GROSS
• 金额: $ 0.02万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7721419
• 关键词: Affinity; Area; Automation; Binding; Binding Proteins; Biophysics; Buffers; Calcium-Binding Proteins; Calmodulin; Computer Retrieval of Information on Scientific Projects Database; DNA-Binding Proteins; Development; Drug Design; Funding; Grant; Institution; Ions; Kinetics; Libraries; Ligands; Mass Spectrum Analysis; Measures; Metals; Methods; Modeling; Peptides; Pharmaceutical Preparations; Proteins; Proteomics; Protocols documentation; Rate; Research; Research Institute; Research Personnel; Resources; Salts; Score; Screening procedure; Solutions; Source; System; Titrations; United States National Institutes of Health; Work; data modeling; drug discovery; fatty acid binding protein; fatty acid-binding proteins; human centrin-2 protein; interest; mutant; novel; protein folding; small molecule; stoichiometry

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Protein-ligand binding and the concomitant conformational change in the protein are of crucial importance in biophysics and drug design. We developed a novel method to quantify protein-ligand interactions in solution by mass spectrometry, titration., and H/D exchange (PLIMSTEX). The approach can determine the conformational change, binding stoichiometry, and affinity in protein-ligand interactions including those that involve small molecules, metal ions, and peptides. Binding consts. obtained by PLIMSTEX for four model protein-ligand systems agree with K values measured by conventional methods. At higher protein concn., the method can be used to determine quickly the binding stoichiometry and possibly the purity of proteins. Taking advantage of concentrating the protein on-column and desalting, we are able to use different concns. of proteins, buffer systems, salts, and pH in the exchange protocol. High picomole quantities of proteins are sufficient, offering significantly better sensitivity than that of NMR and x-ray crystallog. Automation could make PLIMSTEX a high throughput method for library screening, drug discovery, and proteomics. We are continuing the development of a protocol for H/D exchange of proteins. The protocol is directed at an aspect of protein folding that is not being pursued by others; namely the interaction of proteins with metal ions and with other substances such as proteins, peptides, drugs. An area of particular interest is the conformational changes that occur to calcium-binding proteins (e.g., calmodulin, calbinden, and human centrin 2). Another protein system of interest is fatty acid binding protein, an area in which scores of mutants are available from Dr. A. Kleinfeld at Torrey Pines Research Institute. A third system is DNA-binding proteins, and in this area we are working on the application of PLIMSTEX to hTRF2, a small protein for which binding off rates are competitive with rates of H/D exchange. This provides a challenge in modeling PLIMSTEX and kinetic data, and the modeling is underway.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 蛋白质结合和蛋白质中构象变化在生物物理和药物设计中至关重要。 我们开发了一种新的方法，可以通过质谱，滴定和H/D交换（PLIMSTEX）来量化溶液中蛋白质 - 配体的相互作用。 该方法可以决定蛋白质 - 配体相互作用的构象变化，结合化学计量和亲和力，包括涉及小分子，金属离子和肽的相互作用。 绑定常量。通过PLIMSTEX获得的四个模型蛋白质配体系统获得了与常规方法测量的K值一致。 在较高的蛋白质浓度下，该方法可用于快速确定结合化的化学计量和可能的蛋白质纯度。 利用将蛋白质集中在列和脱盐的优势中，我们能够使用不同的浓度。交换协议中的蛋白质，缓冲系统，盐和pH值。 高足菌的蛋白质足够足够，具有比NMR和X射线晶体的敏感性明显更好。 自动化可以使PLIMSTEX成为图书馆筛查，药物发现和蛋白质组学的高吞吐量方法。我们正在继续开发用于H/D交换蛋白质的方案。 该方案针对其他人没有追求的蛋白质折叠方面。即蛋白质与金属离子以及其他物质（例如蛋白质，肽，药物）的相互作用。 特别感兴趣的区域是钙结合蛋白（例如钙调蛋白，卡尔宾登和人类中心蛋白2）发生的构象变化。 感兴趣的另一个蛋白质系统是脂肪酸结合蛋白，该区域在Torrey Pines研究所的A. Kleinfeld博士可获得数分突变体的区域。 第三个系统是DNA结合蛋白，在这一领域，我们正在应用PLIMSTEX应用于HTRF2，这是一种小蛋白，其结合速率与H/D交换速率具有竞争力。 这为建模PLIMSTEX和动力学数据提供了挑战，并且建模正在进行中。