SYNTHESIS, MOLECULAR BIOLOGY, AND BIOANALYTICAL STUDIES OF A FLUORESCENT SEROTONIN LIGAND WITH 5-HT3A IN BULK AND SUPPORTED LIPID BILAYERS
Silva Lopez, Elsa Idaliz
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The first part of this dissertation is based on the synthesis, characterization and binding of a fluorescent ligand (5-HT*) that binds to a recombinant Serotonin type 3 Receptor (5-HT3A). Here a linker and a fluorescent tag was added to the indole nitrogen of 5-Hydroxytryptamine (5-HT), an agonist of 5-HT3A. Next the affinity purified receptor in solution was used and the binding of the ligand to the receptor was studied using luminescence spectroscopy, obtaining a Kd of 83 + 3 nM and a Hill Coefficient n = 3. Also described are results obtained from the binding of this fluorescent ligand to the receptor in supported lipid bilayers using Fluorescence Microscopy and Total Internal Reflection Fluorescence (TIRF) Single Molecule Spectroscopy (SMS). Future efforts will address strategies to reduce the non-specific binding of the ligand to the membranes.The second part of this dissertation involves the study of the effect of cushioned biomimetic lipid membrane assemblies on passivated surfaces containing Bovine Serum Albumin (BSA) and their diffusion properties. This study utilized BSA in its native, denatured, reduced and denatured / reduced states to cover a hydrophilically treated glass surface, followed by lipid bilayer formation. The structure and fluidity of these systems were studied using Fluorescence Microscopy and Fluorescence Recovery after Photobleaching (FRAP). The data obtained show that the BSA state plays a critical role on the deposition, fusion, and diffusion of lipid bilayers. For instances, the presence of denatured or reduced BSA resulted in bilayer formation, but no recovery was observed. In contrast, in the presence of native or denatured and reduced BSA bilayer formation and recovery was observed, indicating the formation of continuous SLB's. The recovery behavior proved to be dependent on the nature of the lipid bilayer and the buffer conditions used.