Harms, MichaelShavlik, Michael2024-03-252024-03-252024-03-25https://hdl.handle.net/1794/29288How do the underlying biophysical properties of proteins dictate the “rules” that govern molecular evolution? Understanding the principles and mechanisms that determine which evolutionary trajectories proteins take is crucial to protecting humans against viral protein evolution and developing therapeutic, custom, drugs through protein engineering. Although many approaches have been developed to investigate the process of protein evolution, a deep understanding of the relationship between sequence space and protein biophysics can alleviate key deficiencies in our knowledge. What is the underlying distribution of functional proteins in sequence? Do specific biophysical properties dictate the interconnectedness of these functional proteins? How does the protein energy landscape change across evolutionary time and how can that inform our understanding of evolution? This dissertation will explore two methods of answering these questions: 1) High-throughput mutagenesis and phenotype characterization to explore sequence space using fluorescent proteins and 2) Ancestral Sequence Reconstruction linked to a biophysical lens using protein energy landscapes.en-USAll Rights Reserved.BiophysicsGFPProtein evolutionSequence spaceElectronic Thesis or Dissertation