NMR studies of membranes proteins & macromolecular systems

膜蛋白的核磁共振研究

• 批准号: 261980-2008
• 负责人: Prosser, Scott
• 金额: $ 3.93万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2008
• 资助国家: 加拿大
• 起止时间: 2008-01-01 至 2009-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=395320
• 关键词: NMR; studies; membranes; proteins; macromolecular

The human genome encodes over 800 G protein-coupled receptors (GPCRs) - a superfamily of membrane proteins responsible for signalling and targetted by over 50% of pharmaceuticals. We propose to develop a protocol for monitoring GPCR membrane protein misfolding and determining the binding site of pharmacophores, using 19F Nuclear Magnetic Resonance (NMR). The protocol entails new strategies for protein expression, new (19F) labeling protocols (including site-directed mutants involving parafluorphenylalanine), and approaches to determine topology and structure through inter-helix distance measurements. Most of this work will be focused on two integral membrane proteins (DAGK and V2R) which are expressed in E coli. The use of fluoro-tryptophan is key in studying misfolding and drug binding sites since tryptophans are typically located at the membrane water interface, and local chemical shift perturbations from pharamcophore binding can thus be registered. 19F shifts and relaxation rates are exquisitely sensitive to local structure and the presence of water- or membrane soluble paramagnetic additives. The combination of paramagnetic shift or rate measurements from both dissolved O2 and water soluble paramagnetic additives provides a simple way to probe local topology (immersion depth, solvent exposure, exposed surface area and local dynamics). Much of the proposed research will be bolstered by extensive membrane model studies and work with soluble proteins (ubiquitin, Mesd, calmodulin, and drkN SH3), some of which is more suited to advanced undergraduate students. Finally, an additional area of research entails new work with paramagnetic nanoparticles (NPs), consisting of gadolinium and fluorine (GdF3) and coated with polyacrylic acid. These NPs give rise to the highest known paramagnetic relaxation rates for MRI contrast agents and are directly applicable as contrast agents for Computed Tomography. Our goal is to develop the GdF3 NPs for targeted imaging and explore their potential as anticancer agents, when appropriately doped with radionuclides.

人类基因组编码超过800 G蛋白偶联受体（gpcr），这是一种膜蛋白超家族，负责信号传导，并被50%以上的药物靶向。我们建议开发一种利用19F核磁共振（NMR）监测GPCR膜蛋白错误折叠和确定药物载体结合位点的方案。该方案需要新的蛋白质表达策略，新的（19F）标记方案（包括涉及多氟苯丙氨酸的位点定向突变），以及通过螺旋间距离测量确定拓扑和结构的方法。大部分工作将集中在大肠杆菌中表达的两种完整膜蛋白（DAGK和V2R）上。荧光色氨酸的使用是研究错误折叠和药物结合位点的关键，因为色氨酸通常位于膜水界面，因此可以记录由药团结合引起的局部化学位移扰动。19F位移和弛豫速率对局部结构和水溶或膜溶顺磁添加剂的存在非常敏感。结合溶解O2和水溶性顺磁添加剂的顺磁位移或速率测量，提供了一种简单的方法来探测局部拓扑（浸入深度、溶剂暴露、暴露表面积和局部动力学）。许多拟议的研究将通过广泛的膜模型研究和可溶性蛋白（泛素，Mesd，钙调素和drkN SH3）的工作得到支持，其中一些更适合高年级本科生。最后，一个额外的研究领域涉及顺磁性纳米颗粒（NPs）的新工作，由钆和氟（GdF3）组成，并涂有聚丙烯酸。这些NPs引起MRI造影剂的最高已知顺磁弛豫率，并直接适用于计算机断层扫描造影剂。我们的目标是开发用于靶向成像的GdF3 NPs，并在适当掺杂放射性核素时探索其作为抗癌剂的潜力。