Probing ion channel macromolecular interactions using fluorescence resonance energy transfer.
Methods Enzymol
; 653: 319-347, 2021.
Article
em En
| MEDLINE
| ID: mdl-34099178
ABSTRACT
Ion channels are macromolecular complexes whose functions are exquisitely tuned by interacting proteins. Fluorescence resonance energy transfer (FRET) is a powerful methodology that is adept at quantifying ion channel protein-protein interactions in living cells. For FRET experiments, the interacting partners are tagged with appropriate donor and acceptor fluorescent proteins. If the fluorescently-labeled molecules are in close proximity, then photoexcitation of the donor results in non-radiative energy transfer to the acceptor, and subsequent fluorescence emission of the acceptor. The stoichiometry of ion channel interactions and their relative binding affinities can be deduced by quantifying both the FRET efficiency and the total number of donors and acceptors in a given cell. In this chapter, we discuss general considerations for FRET analysis of biological interactions, various strategies for estimating FRET efficiencies, and detailed protocols for construction of binding curves and determination of stoichiometry. We focus on implementation of FRET assays using a flow cytometer given its amenability for high-throughput data acquisition, enhanced accessibility, and robust analysis. This versatile methodology permits mechanistic dissection of dynamic changes in ion channel interactions.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteínas
/
Transferência Ressonante de Energia de Fluorescência
Idioma:
En
Revista:
Methods Enzymol
Ano de publicação:
2021
Tipo de documento:
Article
País de afiliação:
Estados Unidos