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Nam Lee

Mentor: John Koland, Ph.D.
Lab Phone: 335-6551

While 3-D structures of extracellular ligand binding and intracellular protein tyrosine kinase domains of the epidermal growth factor receptor (EGFR) have been determined, the structure of the EGFR C-terminal phosphorylation domain remains elusive. The structure and dynamics of this C-terminal phosphorylation domain were investigated by biophysical fluorescence spectroscopic approaches. We first devised a method of site-specifically labeling the extreme C-terminus of the purified recombinant EGFR intracellular domain with a fluorescent probe, which exploited the intein-based protein expression system. Subsequently, time-resolved fluorescence anisotropy measurements were used to quantify the local motion of the C-terminal domain on the nanosecond time scale. Large-scale conformational changes associated with receptor activation/phosphorylation were then assessed by fluorescence resonance energy transfer (FRET). Here, the EGFR C-terminal domain was labeled with a blue fluorescent protein (BFP) module and the catalytic site was labeled with trinitrophenyl-ATP. This allowed an estimation of the distance between the catalytic core and the extreme C-terminus. Both fluorescence anisotropy and FRET measurements showed that upon phosphorylation there was a significant change in conformation of the C-terminal domain, a conformational change that could regulate the catalytic activity of protein tyrosine kinase.