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FRETting about the affinity of bimolecular protein–protein interactions
Author(s) -
Lin Tao,
Scott Brandon L.,
Hoppe Adam D.,
Chakravarty Suvobrata
Publication year - 2018
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.3482
Subject(s) - förster resonance energy transfer , affinities , protein–protein interaction , chemistry , biophysics , dissociation constant , plasma protein binding , fluorescence , biochemistry , biology , receptor , physics , quantum mechanics
F luorescence r esonance e nergy t ransfer (FRET) is a powerful tool to study macromolecular interactions such as protein–protein interactions (PPIs). Fluorescent protein (FP) fusions enable FRET‐based PPI analysis of signaling pathways and molecular structure in living cells. Despite FRET's importance in PPI studies, FRET has seen limited use in quantifying the affinities of PPIs in living cells. Here, we have explored the relationship between FRET efficiency and PPI affinity over a wide range when expressed from a single plasmid system in Escherichia coli. Using live‐cell microscopy and a set of 20 pairs of small interacting proteins, belonging to different structural folds and interaction affinities, we demonstrate that FRET efficiency can reliably measure the dissociation constant (K D ) over a range of m M to nM. A 10‐fold increase in the interaction affinity results in 0.05 unit increase in FRET efficiency, providing sufficient resolution to quantify large affinity differences (> 10‐fold) using live‐cell FRET. This approach provides a rapid and simple strategy for assessment of PPI affinities over a wide range and will have utility for high‐throughput analysis of protein interactions.