INVESTIGATION OF THE STRUCTURE AND FUNCTION OF GLUTAMATE RECEPTOR GLUR2 USING SOLUTION NMR SPECTROSCOPY
Glutamate receptors are the most widespread neurotransmitter binding proteins in the vertebrate nervous system, and have been implicated in a variety of higher brain functions and pathological conditions. A better understanding of the structure and function of these receptors is highly desirable for drug development The first project presented in this dissertation aimed to verify a hypothesis directly relating the activity of a partial agonist with the degree of lobe closure of the soluble ligand binding domain (S1S2) observed upon binding that ligand. This hypothesis was based on analysis of x-ray crystal structures bound to a family of partial agonists, 5substituted willardiines. We used NH RDCs to determine the average solution conformation of S1S2 bound to various ligands, including the 5-substituted willardiines. To obtain the desirable precision of lobe closure calculation, we developed a method by which the structural noise is initially reduced using structure refinement against RDCs measured in multiple media for a single ligand (glutamate). Such refined structures could then be used to reorient lobes of S1S2 based on RDC data measured for any other ligand in a single alignment medium. We were able to calculate the difference in the degree of lobe closure induced by glutamate and any other ligand with precision typically better than +/-0.75 degrees. Results obtained for willardiine partial agonists suggested that the relative orientation of the domains of S1S2 is not the only factor determining efficacies of partial agonists, and that largescale domain motions may be an important factor. The second project presented in this dissertation was to measure fast and slow dynamics of certain types of hydrophobic side chains in GluR2 S1S2 bound to a variety of ligands. This study presented a unique perspective on the protein structure, focusing on the behavior of the hydrophobic cores of S1S2. Slow motions reveal that lobe 2 is organized in such a way that its parts may be able to slide past each other. This organization may be significant for the function of the intact receptor. Slow motions of I712 show that relative domain motions take place for willardiine partial agonists, but not the other tested ligands. These motions are especially pronounced for iodowillardiine, in line with previous experiments. Faster motions provide an opportunity to calculate side chain entropic contribution to binding, and suggest that the structure of S1S2 is specifically adapted to accommodate the only natural agonist, glutamate.
dissertation or thesis