NMR studies of membranes proteins & macromolecular systems

人类基因组编码超过800个G蛋白偶联受体（GPCR）-一个负责信号传导的膜蛋白超家族，并被超过50%的药物靶向。我们建议开发一个协议，用于监测GPCR膜蛋白的错误折叠和确定药效团的结合位点，使用19 F核磁共振（NMR）。该协议需要新的策略，蛋白质表达，新的（19 F）标记协议（包括定点突变，涉及paramorphenylalanine），并通过螺旋间距离测量的方法来确定拓扑结构和结构。本论文的主要工作将集中在两种在大肠杆菌中表达的膜蛋白（DAGK和V2 R）。氟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，因此可以记录来自pharamcophore结合的局部化学位移扰动。19 F位移和弛豫速率对局部结构和水溶性或膜溶性顺磁性添加剂的存在非常敏感。溶解O2和水溶性顺磁添加剂的顺磁位移或速率测量的组合提供了一种简单的方法来探测局部拓扑结构（浸没深度、溶剂暴露、暴露表面积和局部动力学）。大部分拟议的研究将得到广泛的膜模型研究的支持，并与可溶性蛋白（泛素，Mesd，钙调蛋白和drkN SH 3）合作，其中一些更适合高年级本科生。最后，另一个研究领域需要使用顺磁性纳米颗粒（NPs）的新工作，该纳米颗粒由钆和氟（GdF 3）组成，并涂有聚丙烯酸。这些NP产生了MRI造影剂的最高已知顺磁弛豫速率，并且可直接用作计算机断层扫描的造影剂。我们的目标是开发用于靶向成像的GdF 3 NPs，并探索其在适当掺杂放射性核素时作为抗癌剂的潜力。

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.