Single-Molecule Fluorescence Detection of the Epidermal Growth Factor Receptor in Membrane Discs
Name
nihms956744.pdf
Description
Accepted version
Size
606.99 KB
Format
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Checksum (MD5)
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Author(s)
Quinn, Steven D.
Srinivasan, Shwetha
Gordon, Jesse B.
He, Wei
Carraway, Kermit L.
Coleman, Matthew A.
Schlau-Cohen, Gabriela S
Date Issued
March 2018
Journal
Biochemistry
Publisher
American Chemical Society (ACS)
Citation
Quinn, Steven D. et al. "Single-Molecule Fluorescence Detection of the Epidermal Growth Factor Receptor in Membrane Discs." Biochemistry 58, 4 (March 2018): 286–294 © 2018 American Chemical Society
Version
Author's final manuscript
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.
MIT Department
Massachusetts Institute of Technology. Department of Chemistry
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Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use.
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DOI of Published Version
https://doi.org/10.1021/acs.biochem.8b00089
