NMR studies of membranes proteins & macromolecular systems

人类基因组编码超过800G蛋白偶联受体(GPCRs)--一个负责信号传递的膜蛋白超家族，超过50%的药物以此为靶标。我们建议开发一种使用19F核磁共振(核磁共振)来监测GPCR膜蛋白错误折叠和确定药效团结合位置的方法。该协议需要新的蛋白质表达策略，新的(19F)标记协议(包括涉及对氟苯丙氨酸的定点突变)，以及通过螺旋间距测量确定拓扑和结构的方法。大部分工作将集中在在大肠杆菌中表达的两个完整的膜蛋白(DAGK和V2R)上。使用氟色氨酸是研究错误折叠和药物结合部位的关键，因为色氨酸通常位于膜水界面，因此可以登记来自生物质结合的局部化学位移扰动。~(19)F位移和驰豫速率对局部结构和水或膜可溶顺磁性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，可以提供一种简单的方法来探测局部拓扑(浸泡深度、溶剂暴露、暴露表面积和局部动态)。许多拟议的研究将得到广泛的膜模型研究的支持，并研究可溶性蛋白质(泛素、MESD、钙调蛋白和drkN SH3)，其中一些更适合高级本科生。最后，另一个研究领域需要对顺磁性纳米颗粒(NPs)进行新的工作，这种纳米颗粒由Gd和氟(GDF3)组成，并包覆有聚丙烯酸。这些纳米粒子产生了已知的最高的MRI造影剂的顺磁松弛速率，并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GDF3纳米粒子，并探索当适当掺杂放射性核素时，它们作为抗癌剂的潜力。

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.