NMR studies of membranes proteins & macromolecular systems

点击翻译按钮获取中文摘要

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.