DEVELOPMENT OF H/D EXCHANGE FOR PROTEIN BIOPHYSICS

蛋白质生物物理学 H/D 交换的发展

• 批准号: 8168666
• 负责人: MICHAEL L GROSS
• 金额: $ 5.63万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-10 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8168666
• 关键词: Amides; Binding; Biophysics; Complex; Computer Retrieval of Information on Scientific Projects Database; Detection; Development; Funding; Gases; Grant; Institution; Ions; Label; Ligand Binding; Ligands; Literature; Mass Spectrum Analysis; Measures; Metals; Methods; Peptides; Phase; Property; Proteins; Reaction; Research; Research Personnel; Resources; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spectrum Analysis; System; Testing; Titrations; United States National Institutes of Health; 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 interactions by mass spectrometry, titration, and H/D exchange (PLIMSTEX) is a mass spectrometric method for detg. assocn. consts. and binding stoichiometry for interactions of proteins with various ligands, as well as for quantifying the conformational changes assocd. with ligand binding to proteins. The assocn. consts. detd. with PLIMSTEX agree with literature values within a factor of six, establishing its validity for protein interactions involving metal ions, small org. mols., peptides, and proteins. PLIMSTEX provides soln., not gas-phase, properties by taking advantage of ESI and MALDI mass spectrometry to measure accurately the mass of a protein as it undergoes amide H/D exchange. The approach sidesteps the problem of relating gas-phase abundances of the protein or protein-ligand complex ions to their soln. concns. With on-column concn. and desalting, high picomole quantities of proteins are sufficient for reproducible mass detection, and the concn. of the protein can be as low as 10-8 M. It is amenable to different protein/ligand systems in physiol. relevant media. No specially labeled protein or ligand is needed. PLIMSTEX offers minimal perturbation of the binding equil. because it uses no denaturants, no addnl. spectroscopy or reaction probes, and no phys. sepn. of ligand and protein during binding. In this research, we are developing, testing, and extending further the method.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 通过质谱、滴定和 H/D 交换进行蛋白质-配体相互作用 (PLIMSTEX) 是一种用于检测的质谱方法。协会。常量。以及结合化学计量学，用于蛋白质与各种配体的相互作用，以及量化相关的构象变化。配体与蛋白质结合。 协会。常量。已确定。 PLIMSTEX 与文献值的一致性在六倍以内，确定了其对于涉及金属离子、小组织的蛋白质相互作用的有效性。分子、肽和蛋白质。 PLIMSTEX 通过利用 ESI 和 MALDI 质谱法来准确测量蛋白质在进行酰胺 H/D 交换时的质量，从而提供溶液而不是气相特性。 该方法回避了将蛋白质或蛋白质-配体复合离子的气相丰度与其溶液相关联的问题。浓度。 具有柱上浓度。和脱盐，高皮摩尔量的蛋白质足以进行可重复的质量检测，并且浓度。蛋白质的分子量可低至 10-8 M。它适用于生理学中不同的蛋白质/配体系统。相关媒体。 不需要专门标记的蛋白质或配体。 PLIMSTEX 对结合平衡的扰动最小。因为它不使用变性剂，不使用addnl。光谱或反应探针，没有物理。九月配体和蛋白质在结合过程中的变化。 在这项研究中，我们正在开发、测试和进一步扩展该方法。