Electronic Thesis and Dissertation Repository


Master of Science




Dr. David Litchfield


Pin1 is a human protein classified as a peptidyl-prolyl cis/trans isomerase. The protein regulates the conformation of phosphorylated protein substrates by rotating the peptide bond between phosphorylated serine/threonine residues that precede proline residues. Structurally, Pin1 consists of an N-terminal WW domain and a C-terminal PPIase domain. The PPIase domain catalyzes cis/trans isomerization of peptide bonds in substrate proteins that contain the aforementioned consensus motif. We hypothesize that Pin1 binding is positively impacted when two phospho-acceptor sites on peptides derived from mitotic phosphatase CDC25C, a known Pin1-interacting protein, are phosphorylated. Using nuclear magnetic resonance and fluorescence polarization, binding affinities of CDC25C peptides to Pin1 were calculated. The results indicate that doubly-phosphorylated peptides bound to Pin1 have lower dissociation constants and consequently greater binding affinities, than complexes containing non- or singly-phosphorylated peptides, at the equivalent residues. This suggests that Pin1 has two independent phospho-binding sites that when bound, increase substrate binding affinity.