42859-AC4
Determination of Ligand Binding Epitopes Using NMR Diffusion Measurements
During the past year of this grant we continued to make good progress on this project. We have completed a study on the binding of R- and S-enantiomers of propranolol to the serum protein alpha1-acid glycoprotein (AGP) and that work is in press in the Journal of Physical Chemistry. About 45% of the mass of AGP is made up by carbohydrate and no high resolution structure of AGP is available. Experiments in progress have as their goal understanding of the binding of the smaller and achiral drug lidocaine at the same binding site. Our preliminary results measured using NOESY, diffusion and saturation transfer difference (STD) experiments suggest that the smaller but achiral lidocaine molecule appears to have a similar binding motif to propranolol. However, experiments performed as a function of ligand concentration reveal evidence on non-specific binding. Non-specific binding is a common problem in studies of ligand-protein binding. We are developing a strategy for factoring out non-specific binding from the results of STD NMR data sets. This work has the potential to be of significant impact for biochemical studies. We expect these experiments to be completed in the next few months and hope to have a paper submitted on this work by the beginning of the year.
Our group has also used these NMR methods to probe intermolecular interactions important in separations. The diffusion, NOESY and STD experiments are especially useful for understanding interactions involved in chiral micellar electrokinetic chromatography (MEKC) separations involving molecular micelles. During the remaining period of this grant, we plan to extend our previous work the examination of interactions of analytes in solution with solid phase chromatographic supports. This can be done using to high-resolution magic angle spinning (HR-MAS) NMR to reduce the linewidth by averaging out the magnetic susceptibility differences introduced by the particles. These HR-MAS experiments can be coupled with NMR diffusion and STD measurements to probe the molecular level interactions important in chromatographic separations.
During the past year this grant assisted in the support and development of a graduate student, Jennifer Cruz. Jennifer is a Ph.D. candidate in the department of chemistry at UCR who has just begun her fourth year of graduate school. The previous Ph.D. student supported by this grant, Dr. Bridget Becker, is now a research scientist at Merck in
