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

TY - JOUR

T1 - Single-Molecule Fluorescence Detection of the Epidermal Growth Factor Receptor in Membrane Discs

AU - Quinn, Steven D

AU - Srinivasan, Shwetha

AU - Gordon, Jesse B

AU - He, Wei

AU - Carraway, Kermit L

AU - Coleman, Matthew A

AU - Schlau-Cohen, Gabriela S

PY - 2019/1/29

Y1 - 2019/1/29

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=85053607478&partnerID=8YFLogxK

U2 - 10.1021/acs.biochem.8b00089

DO - 10.1021/acs.biochem.8b00089

M3 - Article

C2 - 29553754

SN - 0006-2960

VL - 58

SP - 286

EP - 294

JO - Biochemistry

JF - Biochemistry

IS - 4

ER -