Single-Molecule Fluorescence Detection of the Epidermal Growth Factor Receptor in Membrane Discs

Steven D. Quinn, Shwetha Srinivasan, Jesse B. Gordon, Wei He, Kermit L Carraway, Matthew A Coleman, Gabriela S. Schlau-Cohen

Research output: Contribution to journalArticle

Abstract

The epidermal growth factor receptor (EGFR) is critical to normal cellular signaling pathways. Moreover, it has been implicated in a range of pathologies, including cancer. As a result, it is the primary target of many anticancer drugs. One limitation to the design and development of these drugs has been the lack of molecular-level information about the interactions and conformational dynamics of EGFR. To overcome this limitation, this work reports the construction and characterization of functional, fluorescently labeled, and full-length EGFR in model membrane nanolipoprotein particles (NLPs) for in vitro fluorescence studies. To demonstrate the utility of the system, we investigate ATP-EGFR interactions. We observe that ATP binds at the catalytic site providing a means to measure a range of distances between the catalytic site and the C-terminus via Förster resonance energy transfer (FRET). These ATP-based experiments suggest a range of conformations of the C-terminus that may be a function of the phosphorylation state for EGFR. This work is a proof-of-principle demonstration of single-molecule studies as a noncrystallographic assay for EGFR interactions in real-time and under near-physiological conditions. The diverse nature of EGFR interactions means that new tools at the molecular level have the potential to significantly enhance our understanding of receptor pathology and are of utmost importance for cancer-related drug discovery.

Original languageEnglish (US)
JournalBiochemistry
DOIs
StateAccepted/In press - Jan 1 2018

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Epidermal Growth Factor Receptor
Fluorescence
Membranes
Molecules
Adenosine Triphosphate
Pathology
Catalytic Domain
Cell signaling
Phosphorylation
Drug Design
Energy Transfer
Drug Discovery
Pharmaceutical Preparations
Energy transfer
Conformations
Assays
Neoplasms
Demonstrations
Experiments

ASJC Scopus subject areas

  • Biochemistry

Cite this

Single-Molecule Fluorescence Detection of the Epidermal Growth Factor Receptor in Membrane Discs. / Quinn, Steven D.; Srinivasan, Shwetha; Gordon, Jesse B.; He, Wei; Carraway, Kermit L; Coleman, Matthew A; Schlau-Cohen, Gabriela S.

In: Biochemistry, 01.01.2018.

Research output: Contribution to journalArticle

Quinn, Steven D. ; Srinivasan, Shwetha ; Gordon, Jesse B. ; He, Wei ; Carraway, Kermit L ; Coleman, Matthew A ; Schlau-Cohen, Gabriela S. / Single-Molecule Fluorescence Detection of the Epidermal Growth Factor Receptor in Membrane Discs. In: Biochemistry. 2018.
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