Date of Award
Fall 9-5-2025
Degree Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Chemistry
First Advisor
Joerg Zimmermann
Abstract
Engineered antibodies (Abs) have found applications in disparate fields such as enzyme catalysis and medicinal therapeutics because of their exquisite binding affinity and specificity. To engineer antibodies for these purposes, a firm understanding of the underlying mechanisms that control Ab affinity and specificity is needed, so these properties can be tightly controlled and adapted to the application at hand. My research characterized the binding site of the anti-fluorescein Ab 4-4-20 using an array of biophysical techniques and gives insight into the binding site interactions that give rise to both affinity and specificity. We utilized Fab fragments of the anti-fluorescein Ab 4-4-20 as a model system to investigate the binding site of mature Abs. Site-directed mutagenesis was used to insert eight point-mutations into the Ab binding site at key amino acid residues that directly interacted with fluorescein in the binding pocket. The inherent fluorescence of the fluorescein antigen was utilized in a label-free fluorescence titration used to measure the dissociation constant Kd of fluorescein-Ab binding. Binding specificity was also assessed using the same technique but with a chlorine-substituted fluorescein analog. Binding was further assessed by fluorescence lifetime measurements of Ab-bound fluorescein using time-correlated single photon counting (TCSPC). Finally, the structural stability of the point mutants was determined using thermal melt curves with SYPRO® Orange (SyO) as a reporter dye. Additionally, we are interested in comparing the binding behavior of mature and germline Abs. We are optimizing the papain digestion of IgG Abs to FAB fragments which we will use with our inherent fluorescence titration technique to characterize the binding of germline and mature Ab pairs that also bind to fluorescein. SyO is a zwitterionic dye that is used for protein gel staining, thermal melt assays of proteins, and as a marker for misfolded proteins. However, while widely utilized, much of SyO’s photophysics remains unexplored. We studied the effect of pH on the photophysical properties of SyO in aqueous solution and found two well-defined transitions in the 0 to 10 pH range between three SyO species with distinct absorption and fluorescence properties. The aggregation behavior of the three pH-dependent species of SyO were characterized by their absorbance and fluorescence spectra. The aggregation behavior of SyO at neutral pH and aqueous solution was found to be essential to the utility of SyO as a reporter dye in protein assays. Furthermore, SyO-based assays need to be optimized for each individual protein system and this optimization process can be challenging because the mechanisms by which SyO fluorescence is increased through protein interactions are poorly understood. We characterized the steady-state and time-correlated fluorescence of SyO interacting with hen egg-white lysozyme (HEL), bovine alpha-lactalbumin (aLac), saccharomyces cerevisiae invertase (Inv), and bovine serum albumin (BSA) at a range of temperatures to understand the temperature-dependent mechanisms that lead to the enhancement of SyO fluorescence when it interacts with the model proteins.
Recommended Citation
Baxter, Claire, "Analysis of Antibody Affinity Maturation and Thermal Stability of Proteins Using Photophysical Approaches" (2025). Dissertations. 4209.
https://ecommons.luc.edu/luc_diss/4209
