Home List of Titles Creation and biophysical characterization of a high-affinity, monomeric EGF receptor ectodomain using fluorescent proteins
Please use this identifier to cite or link to this item: http://hdl.handle.net/1959.3/92865
- Creation and biophysical characterization of a high-affinity, monomeric EGF receptor ectodomain using fluorescent proteins
- Kozer, Noga; Henderson, Christine; Bailey, Michael F.; Rothacker, Julie; Nice, Edouard C.; Burgess, Anthony W.; Clayton, Andrew H. A.
- X-ray structural studies revealed two conformations of the epidermal growth factor receptor (EGFR) ectodomain (ECD): a compact, tethered conformation in the absence of EGF and an untethered or extended conformation in the presence of EGF. An EGFR-ECD derivative with a monomeric red fluorescent protein (mRFP) at the N-terminus and an enhanced green fluorescent protein (eGFP) at the C-terminus (dual-tag-EGFR-ECD) was created and characterized. The dual-tag-EGFR-ECD construct was shown to have high affinity (nanomolar range) for both EGF and EGFR monoclonal antibody (mAb528). The dual-tag-EGFR-ECD was further characterized by fluorescence-detected analytical ultracentrifugation, lifetime FRET, and fluorescence anisotropy. We found no evidence of a tethered unliganded conformation, nor did we observe a large shape change upon ligand binding as predicted by the crystal models. Increases in steady-state anisotropy upon binding of EGF to the dual-tag-EGFR-ECD were observed and interpreted as changes in the protein flexibility and dynamics. We conclude the fluorescent protein tags perturb the EGFR-ECD structure, making it extended with a 50-fold higher affinity for EGF relative to that of the nontagged EGFR-ECD.
- Publication type
- Journal article
- Research centre
- Swinburne University of Technology. Faculty of Engineering and Industrial Sciences. Centre for Micro-Photonics
- Biochemistry, Vol. 49, no. 35 (2010), pp. 7459-7466
- Publication year
- FOR Code(s)
- 0601 Biochemistry and Cell Biology; 1101 Medical Biochemistry and Metabolomics
- ECD; Ectodomain; EGF; Epidermal growth factor; Fluorescent proteins
- American Chemical Society
- Publisher URL
- Copyright © 2010 American Chemical Society. The American Chemical Society does not allow institutions to archive either the accepted manuscript or the published version of the article.
- Peer reviewed