Date of Award
Doctor of Philosophy
Small molecules are known to play critical role in understanding most biological mechanisms of cells and organisms. Some examples, such as RNAs, peptides and drug molecules, etc., work by modulating cellular function, but with unknown modes. In most cases, these actions involve the small molecule interacting with proteins serving various functions. In recent years, much effort has been made in the investigation of interactions between small molecules (ligands) and target proteins. In our laboratory, a new technique termed Dynamic Isoelectric focusing Anisotropy Binding Ligand Assay (DIABLA) is being in collaboration with the Tolley Laboratory (SIU) developed to fulfill this task. In this technique, a protein mixture is separated within the capillary using dynamic isoelectric focusing, while a specific small molecule is evenly distributed throughout the capillary. Fluorescence anisotropy is then used to identify target proteins that bind with the ligand. In our research, emphasis has been put on evaluating optimum detection conditions for the fluorescence anisotropy aspects of the measurement. Fluorescence anisotropy has been proven to be an effective and powerful tool in evaluating ligand-protein interactions. In our studies, various protein-ligand systems are investigated, especially inhibitor-cyclooxygenase (COX) systems which include naproxen-COX system, ibuprofen-COX system, resveratrol-COX system and COX inhibitor II-COX system. Other systems include biotin-streptavidin system and progesterone-progesterone receptor system. Several fundamental parameters (concentration, pH, etc.) that affect the detection of fluorescence anisotropy measurement are evaluated. In addition, non-specific binding of the ligands with BSA was also tested as a comparison to specific binding of ligand-COX. By optimizing the binding conditions, the detection limit of using fluorescence anisotropy technique was found to be as low as nanomolar concentrations, which is much improved compared to the current literature reported micromolar regime. A binding curve representing the anisotropy's value as a function of protein concentration was constructed experimentally for each study system. On another study, mathematical calculation of the binding curve was also carried out by Wolfram Mathematica for prediction of the binding curve as well as estimation of the dissociation constant (Kd). By simply curve fitting experimental data to our simulated binding curve, with known ligand concentration, the dissociation constant (Kd) can be obtained with very high accuracy relative to current reported value. Isoelectric focusing coupled fluorescence anisotropy was also performed on the laboratory built system to test the validation of DIABLA. Three standard dyes, rose bengal, erythrosin B and Ru(bpy)3 were used for calibration of the in-laboratory built instrument. Fluorescence measurements were performed in both Horiba Jobin Yvon fluorimeter and our in-laboratory built DIABLA equipment by Cecil Bailey. Good correspondence of data acquired by DIABLA equipment and Horiba fluoremeter was successfully obtained, which proves the validation of DIABLA. Ongoing research is focusing on investigation of the standard dyes as well as some protein mixtures in capillary using DIABLA equipment. In another study, in investigation of inhibitor-COX system, fluorescence properties of most inhibitors were tested for further applications. Fluorescence excitation and emission spectra, fluorescence quantum yield, as well as fluorescence lifetimes were tested with the inhibitors dissolved in both ethanol and water. The difference of fluorescence properties observed in different solvents revealed the solvent effects as well as some possible intramolecular transitions or intermolecular interactions, such as internal charge transfer (ICT) and molecule aggregations.
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