DEVELOPMENT OF H/D EXCHANGE FOR PROTEIN BIOPHYSICS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 蛋白质-配体相互作用的质谱学、滴定和氢/氢交换(PLIMSTEX)是一种用于检测蛋白质-配体相互作用的质谱学方法。阿瑟恩。康斯特斯。以及结合化学计量学，用于蛋白质与各种配体的相互作用，以及用于量化相关的构象变化。与蛋白质结合的配基。那个混血儿。康斯特斯。特德。PLIMSTEX与文献值在六倍的范围内一致，确立了其对涉及金属离子的蛋白质相互作用的有效性，小组织。摩尔、多肽和蛋白质。PLIMSTEX通过利用ESI和MALDI质谱学来精确测量蛋白质在进行酰胺H/D交换时的质量，从而提供了溶液而不是气相的性质。该方法回避了将蛋白质或蛋白质-配体络合物离子的气相丰度与其溶液相关联的问题。康奈尔人。使用列上的Conn。而脱盐，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。可低至10~(-8)M，可适应不同的蛋白质/配体系统。相关媒体。不需要特殊标记的蛋白质或配体。PLIMSTEX提供对结合Equil的最小扰动。因为它不使用变性剂，也不添加任何添加剂。光谱学或反应探头，没有物理化学物质。SEPN。在结合过程中配体和蛋白质之间的相互作用。在本研究中，我们正在开发、测试和进一步扩展该方法。

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.