New mass spectrometry tools for characterizing protein-ligand interactions

用于表征蛋白质-配体相互作用的新质谱工具

• 批准号: 205047-2013
• 负责人: Klassen, John
• 金额: $ 5.03万
• 依托单位: University of Alberta
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2015
• 资助国家: 加拿大
• 起止时间: 2015-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=570036
• 关键词: New; mass; spectrometry; tools; characterizing

The objectives of this proposal are the development and application of new mass spectrometry (MS) methods to detect and characterize protein-ligand interactions. The proposed research will focus on three areas: (i) Discovery and characterization of host-cell receptors. The interactions of pathogen-generated proteins with carbohydrate receptors on the surfaces of epithelial cells are critical events in many infectious diseases. However, the identification of the functional receptors remains a significant challenge. The goal of the planned research is to develop a novel catch-and-release electrospray ionization (ESI) MS assay for detecting specific interactions between pathogen-generated proteins and their carbohydrate receptors in cell membranes. Notably, the assay will employ direct ESI-MS analysis of aqueous solutions of receptor-binding proteins and receptors, which are incorporated into nanodiscs (NDs). Application of this assay to NDs prepared from lipids extracted from cell membranes will allow for the discovery of human receptors of a variety of bacterial and viral proteins. (ii) Quantifying ligand binding to large protein assemblies. The direct ESI-MS binding assay is an established tool for quantifying protein-ligand binding in solution. However, the requirement that the free and ligand-bound protein ions can be directly detected and accurately quantified imposes limitations on the interactions that can be investigated. The goal of this project is to develop new ESI-MS assays to quantify ligand interactions with large protein complexes, such as virus particles, in vitro. (iii) Elucidating the molecular origin of hydrophobic protein-ligand interactions. Hydrophobic bonding plays important roles in many biochemical processes, such as protein folding and non-covalent association. However, a quantitative description of the underlying forces is lacking. The goal of the proposed research is to develop a more complete description of the interplay between intrinsic interactions and solvent effects in hydrophobic protein-ligand interactions.

本提案的目标是开发和应用新的质谱（MS）方法来检测和表征蛋白质-配体相互作用。拟议的研究将集中在三个领域： (i)宿主细胞受体的发现和表征。病原体产生的蛋白质与上皮细胞表面上的碳水化合物受体的相互作用是许多感染性疾病中的关键事件。然而，功能性受体的鉴定仍然是一个重大的挑战。计划研究的目标是开发一种新的捕获和释放电喷雾电离（ESI）MS检测方法，用于检测病原体产生的蛋白质与细胞膜中碳水化合物受体之间的特异性相互作用。值得注意的是，该测定将采用受体结合蛋白和受体的水溶液的直接ESI-MS分析，所述受体结合蛋白和受体被掺入纳米盘（ND）中。将该测定应用于从细胞膜提取的脂质制备的ND将允许发现各种细菌和病毒蛋白的人受体。 (ii)定量配体与大蛋白组装体的结合。直接ESI-MS结合试验是用于定量溶液中蛋白质-配体结合的既定工具。然而，自由和配体结合的蛋白质离子可以被直接检测和准确定量的要求，可以研究的相互作用施加了限制。该项目的目标是开发新的ESI-MS分析，以量化配体与大蛋白复合物（如病毒颗粒）的体外相互作用。 (iii)阐明疏水蛋白质-配体相互作用的分子起源。疏水键在许多生物化学过程中起着重要的作用，如蛋白质折叠和非共价结合。然而，缺乏对潜在力量的定量描述。该研究的目的是建立一个更完整的描述疏水蛋白质-配体相互作用中内在相互作用和溶剂效应之间的相互作用。