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201289 https://hdl.handle.net/2286/R.2.N.201289 http://rightsstatements.org/vocab/InC/1.0/ All Rights Reserved 2025 2027-05-01T17:44:23 142 pages Doctoral Dissertation Academic theses en Stahl, Erik Mills, Jeremy H Ghirlanda, Giovanna Wang, Xu Arizona State University Partial requirement for: Ph.D., Arizona State University, 2025 Field of study: Biochemistry Fluorescence-based measurements can enhance understanding of biological systems, including antibody-antigen interactions. Generating fluorescent antibodies that are responsive to antigen binding could lead to the development of new diagnostic tools. Previous research explored the development of new fluorescent proteins by using genetically encoded fluorescent non-canonical amino acids (fNCAAs), specifically L-(7-hydroxycoumarin-4-yl)ethylglycine (7-HCAA). In previous research, 7-HCAA was incorporated within the binding site of the antibody 5c8, giving a variant that exhibited a fluorescence increase upon binding with its antigen, CD40L. Structural analysis demonstrates that conformational changes occur in the constant domain upon binding. Observing this, incorporating 7-HCAA within the flexible hinge region connecting the two domains could decouple the fluorescence abilities from the antigen binding site. Success in this endeavor could pave the way for a general method of converting antibodies into fluorescent sensors for their antigens. To test this, 7-HCAA is genetically encoded in the hinge region of 5c8. One mutant, T89X, demonstrates a ~28% increase in fluorescence upon CD40L binding, but only at the excitation wavelength of the deprotonated form of 7-HCAA. Computational modeling using Rosetta suggests that 7-HCAA likely forms hydrogen bonds with main-chain carbonyls; these interactions are likely dissolved by allosteric changes that occur upon binding. Coupling modeling with mutational analysis allows for the discovery of residues that play a role in the modulation of fluorescence between states. This mutational analysis also provided for an optimized 5c8 construct, demonstrating a change of fluorescence of ~48%. This was due to the mutations that result in a more basic protein environment, enabling for more deprotonation. Combining mutations to optimize the fluorescence change shows one set of mutations with a ~63% increase. Unfortunately, applying these mutations to anti-lysozyme shows no change in fluorescence. This is due to the lack of conformational change in anti-lysozyme that is seen in 5c8. Even though the set of mutations were not as applicable as believed, this new fluorescent antibody can serve as a scaffold to detect other proteins, utilizing CDR loop modeling to transfer binding residues to 5c8. Furthermore, studies at modifying the protein environment around 7-HCAA allow for the further fine-tuning of its fluorescence capabilities. Biochemistry Antibody Fluorescence Non-canonical Amino Acids Proteins Rational Design and Optimization of an Antibody-Based Fluorescent Sensor