| Detection and identification of intermolecular interactions are important process of drug discovery and design, because biological functions are usually performed by intermolecular interactions that widely exist among biomacromolecules or between biomacromolecules and small molecule ligands. Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for intermolecular interaction detection and ligands screening, by which intermolecular interactions can be detected at atomic resolution and ps to s time scale under nearly physiological conditions. Therefore, NMR has been increasingly appreciated in academic and industrial application. Generally, NMR techniques for intermolecular interaction detection can be classified into two categories:receptor-detected approaches and ligand-detected approaches. Receptor-detected approaches can provide structure and dynamics information of the binding sites in receptor, but these methods often need milligram quantities of isotope enriched protein. Compared with the receptor-detected approaches, ligand-detected methods can’t provide structure information of the binding site, but these approaches need much smaller amount of unlabelled receptor and are not limited by the receptor size, and can be widely used for drug screening. This dissertation aims to develop new NMR methods for the detection of intermolecular interactions.1. Development of RD-WaterLOGSY:WaterLOGSY experiment is one of the most widely used ligand screening approach, in which the large bulk water magnetization is partially transferred via the protein-ligand complex to the free ligand in a selective manner and so small molecule ligands of the target can be identified rapidly. In WaterLOGSY type methods, long selective pulse or combined pulse are used to achieve water magnetization selectieve excitation or invertion, which result in complex experimental setup and signal loss. Radiation damping is a well-known phenomenon associated with strong magnetization and can cause line-shape distortion and other artifacts in NMR experiment, especially when aqueous sample is measured in high field NMR spectrometer. Radiation damping can be used to achieve selective invertion of the water resonance. Here, we propose an improved WaterLOGSY experiment by utilizing the radiation damping effect to achieve selective inversion of the water resonance (RD-WaterLOGSY). The new method is demonstrated to be sensitive, robust, and convenient for academic and industrial application in ligand screening.2. Implementation of1H-14N NMR for studing protein-ligand interaction:At natural abundance, as much as99.6%of the nitrogens in proteins are14N, however, its fast quadrupole relaxation effects (spin quantum number1=1) severely restrict its application in solution NMR. It was found that trimethyl modified14N usually have narrow line width and can be detected selectively with high sensitivity, because of its symmetric electron cloud around. Here,1H-14N HSQC experiment has been implemented for selective detection of yeast iso-1-cytochrome c, one of the important trimethyllysine protein at natural abundance, and we found that1H-14N correlation spectroscopy can be used to probe the conformation change of the protein at natural abundance when changing the pH or adding the ligands. The method have the potential to be used as a normal probe for proteins with trimethyl modification lysine which is widespread in eukaryotic systems.3. Utilization of!H-14N NMR for probing trimethyl modification of protein:In order to further expand the application of1H-14N correlation spectroscopy, and to make it usefull in normal natural abundance protein, we achieved trimethyl modification of GB1with iodomethane. It was found that side chains N of all six lysines in GB1have been effectively trimethyl modified without changing its3D structure. This indicates that1H-14N NMR can be used for identifying trimethyl modification of proteins and possibly for studing the structural change, interaction and dynamics of proteins at natural abundance. |
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