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195234 https://hdl.handle.net/2286/R.2.N.195234 http://rightsstatements.org/vocab/InC/1.0/ All Rights Reserved 2024 128 pages Doctoral Dissertation Academic theses Text eng Rahman, Mohammad Imtiazur Ghirlanda, Giovanna Borges, Chad Mills, Jeremy Arizona State University Partial requirement for: Ph.D., Arizona State University, 2024 Field of study: Biochemistry Proteins are composed of various arrangements of a common set of amino acids. These arrangements allow proteins to adopt distinct structures and perform specific functions. Metalloproteins, such as cytochrome b562 with an organometallic cofactor, have been studied as mimics of hydrogenase enzymes, showing favorable reaction conditions for hydrogen production. Rational design has revealed that the intrinsic reactivity of the organometallic center is modulated by the mutating protein scaffold. However, studying the effects of mutations on these proteins is challenging due to difficulties in expressing them as holoproteins and the lack of an assay for screening the mutants for hydrogen production. The first chapter presents a protocol for directly expressing proteins with Co-protoporphyrin IX (CoPPIX) to harness its catalytic activity in hydrogen production. Using a high-throughput cell-based assay, the directed evolution of Co-cytochrome b562 is investigated by creating and screening a mutant library. However, this assay platform is incompatible with high-throughput screening due to its low sensitivity, necessitating the development of an alternative platform. A low-throughput assay and experimental framework is proposed to study the directed evolution of metalloproteins. The second chapter explores a de novo-designed cofactor binding peptide (M1) to elucidate biomolecular condensation in living cells. The goal is to understand the physical principles and underlying characteristics of the condensation by M1. Based on a previous discovery where M1-CoPPIX showed hydrogel properties at high concentrations, here, it demonstrates liquid-liquid phase separation through complex coacervation in vitro. E. coli BL21 overexpressing M1 show foci formation at the cell poles. Finally, in a collaborative project, a naturally occurring WW domain protein, N21, was investigated to restore its ligand binding capabilities. This study demonstrates that the structure and dynamics-based design can restore ligand binding functions in N21. Overall, this dissertation employs protein engineering to understand the biophysical characteristics and functions of proteins. Biochemistry Bioengineering Biophysics Biomolecular Condensation Directed Evolution Hydrogel Ligand-binding Liquid-Liquid Phase Separation Metalloproteins Engineering Proteins to Elucidate Biophysical Characteristics and Function