Total Internal Reflection - Fluorescence Correlation Spectroscopy (TIR-FCS): Application to the Study of Ligand - Receptor Interactions

Ligand-receptor interactions are an integral part of cellular processes. Fully understanding these processes requires that the thermodynamic and kinetic parameters of ligand-receptor interactions be measured. Total internal reflection fluorescence microscopy combined with fluorescence correlation spectroscopy (TIR-FCS) can be used to characterize the interactions between a fluorescent ligand and surface-associated receptors. The overall objective of this work was to develop TIR-FCS so as to ease its implementation, and expand its application to the study of complex ligand-receptor systems. Theoretical models describing ligand-receptor interactions measured by TIR-FCS depend on numerous parameters, which complicate the identification of optimal experimental conditions. Criteria, that, if satisfied, would yield autocorrelation curves containing significant information about the kinetics, were defined. Parameter space was systematically explored to identify experimental conditions that satisfy the criteria. Theoretical work has indicated that TIR-FCS curves contain information about nonfluorescent species that associate with receptors and, thereby, alter the interaction of receptors and fluorescent reporter-ligands. Two nonfluorescent species were defined: 1) nonfluorescent effectors (NE) allosterically enhance or inhibit the binding of fluorescent ligand and receptors; while 2) nonfluorescent competitors (NC) compete with fluorescent species for receptors. To test these theoretical predictions, work was conducted to establish systems consisting of a NE and NC. The pregnane X receptor (PXR), a transcription factor, peptides derived from co-activator and co-repressor proteins and a PXR ligand, rifampicin, were chosen to test NE theory. It was found that, contrary to the existing model of PXR action, rifampicin fails to allosterically enhance and reduce PXR's affinity for co-activator and co-repressors, respectively. The biological significance of these results is discussed. These findings preclude the system from being used to test NE theory. As co-activators and co-repressors compete for PXR, the system can be used to test NC theory. An IgG and Fc receptor, FcγRII, were initially chosen to test NC theory. Peptides derived from the antibody binding site on FcγRII were tested to identify those that compete with soluble FcγRII for surface-bound IgG. A poorly soluble peptide that performs this function was identified. An improvement in solubility is required if the system is to be used to test NC theory.Doctor of Philosoph