Creation and biophysical characterization of a high-affinity, monomeric EGF receptor ectodomain using fluorescent proteins.
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.
Kozer
Noga
N
Ludwig Institute for Cancer Research, P.O. Box 2008, Royal Melbourne Hospital, Victoria 3050, Australia.
Henderson
Christine
C
Bailey
Michael F
MF
Rothacker
Julie
J
Nice
Edouard C
EC
Burgess
Anthony W
AW
Clayton
Andrew H A
AH
D002478:Cells, Cultured; D019281:Dimerization; D004815:Epidermal Growth Factor / Q000737:chemistry / Q000378:metabolism; D066246:ErbB Receptors / Q000737:chemistry* / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D014162:Transfection
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004815:Epidermal Growth Factor; D066246:ErbB Receptors