Characterizing Ligand-Protein Interactions by Ligand-Detected Nuclear Magnetic Resonance (NMR) Methods
- Author(s): Cruz, Jennifer
- Advisor(s): Larive, Cynthia
- et al.
The study of ligand-protein interactions is important for understanding the biochemical basis of many diseases and provides an opportunity for the development of pharmaceutical compounds with agonistic or inhibitory activity. Biochemists and medicinal chemists often use structure-activity relationships (SARs) to gain insights into how a ligand's molecular structure is related to specific interactions with a receptor, such as a protein. Nuclear magnetic resonance (NMR) spectroscopy is an ideal technique for probing ligand-protein interactions because a single analysis can yield rich structural information without destroying the sample, it is universal, and analyte derivatization is unnecessary. NMR ligand epitope maps gives information on the ligand protons that interact most strongly with the protein and can provide some information about the ligand's orientation at the binding site. Ligand-detected NMR methods such as nuclear Overhauser spectroscopy (NOESY), diffusion NMR, and saturation transfer difference (STD) can be used to generate ligand epitope maps. The goal of this dissertation is to evaluate the efficiency and reliability by which epitope maps of ligand-protein interactions are determined, advancing the study of fundamental biochemical interactions.
The protein investigated in this research is the human plasma protein, á1-acid glycoprotein (AGP). AGP is heavily glycosylated with about 45% of its mass composed of carbohydrate. AGP binds to a variety of small basic ligands and is known to play an important role in inflammatory processes.
Epitope mapping results will be presented and compared for the binding of the drugs lidocaine and disopyramide with AGP. Lidocaine is an anesthetic drug with a weak binding affinity for AGP, while disopyramide is a chiral drug with stronger binding affinity. The enantiomers of disopyramide were isolated using an AGP chiral stationary phase and the AGP epitope maps of each enantiomer were characterized. Analytical challenges such as effects of nonspecific binding on STD epitope maps and designing the best practice for setting the ligand-protein concentration ratios are also addressed.
AGP is utilized as a chiral chromatographic phase. Preliminary work using high resolution-magic angle spinning (HR-MAS) to characterize the interactions of lidocaine with the AGP stationary phase is presented.