Poster #103, UCSF
Investigating the Effects of Single Mutations on the Stability of a De Novo Designed Homodimeric Protein
Mentors: Angelika Arada; William DeGrado, PhD
Proteins are essential macromolecules, built from amino acids, that adopt a wide variety of structures to carry out nearly all biological processes. Our lab designs de novo proteins to better understand protein functions, how protein structures influence function and to develop proteins with improved or novel properties. This project aims to clone a series of single mutations at the dimer interface of a de novo designed homodimeric protein to investigate effects on dimerization. Based on the location of each mutation, properties of the amino acids, and AlphaFold3 confidence scores we developed a hypothesis predicting which mutations would have the greatest destabilizing effect on the homodimeric protein. We then performed gibson assembly, bacterial transformation, and protein expression to grow Escherichia Coli (E.coli) cells that expressed our protein mutants.The proteins were purified via Ni-NTA affinity chromatography and analyzed by size exclusion chromatography (SEC) to determine whether the mutations affected protein dimerization. Sanger sequencing confirmed successful insertion of the gene of interest into the bacterial plasmid via Gibson Assembly, and SDS-PAGE analysis, verified that all five mutants were successfully expressed and purified. We expect the SEC to show differences in dimerization across the different mutants. These findings will clarify the molecular basis of protein dimerization and inform future studies in protein engineering.