Ligand-activated BRET9 imaging for measuring protein-protein interactions in living mice.

Chem Commun (Camb) 2019 Dec;56(2):281-284

Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba 305-8569, Japan.

Bioluminescence resonance energy transfer (BRET) is a commonly used assay system for studying protein-protein interactions and protein folding in vivo. Conventional BRET systems have solely depended on an overlap of the energy donor and acceptor spectra. In this study, we engineered a conceptually unique ligand-activatable BRET system (termed BRET9), where a full-length Artificial Luciferase variant 23 (ALuc23), acting as the energy donor, is sandwiched between a protein pair of interest, FRB and FKBP12, and linked to a fluorescent protein as the energy acceptor. A specific ligand, rapamycin, then activates inter- and intramolecular interactions of FRB and FKBP12, which develop molecular strain in the sandwiched ALuc23 to accelerate further folding. We found that this system greatly enhanced both the total bioluminescence spectrum and the BRET signal in the far-red (FR) region. We characterized the molecular construct by studying 18 different designs categorized into four groups. The best BRET system design allowed an approximately 5-fold enhancement of the bioluminescence intensities in the FR region. This new BRET system provides a robust ligand-activatable platform that efficiently reports FR bioluminescence signals in cells and living animal models.

Download full-text PDF

Source
http://dx.doi.org/10.1039/c9cc07634dDOI Listing
December 2019

Publication Analysis

Top Keywords

bret system
12
energy donor
8
protein-protein interactions
8
frb fkbp12
8
bret
6
system
5
fkbp12 develop
4
interactions frb
4
intramolecular interactions
4
rapamycin activates
4
activates inter-
4
inter- intramolecular
4
develop molecular
4
strain sandwiched
4
system greatly
4
greatly enhanced
4
enhanced total
4
folding system
4
accelerate folding
4
sandwiched aluc23
4

References

(Supplied by CrossRef)

Vinayagam et al.
Nat. Methods 2014

Michnick et al.
Curr. Opin. Struct. Biol. 2001

Dragulescu-Andrasi et al.
Proc. Natl. Acad. Sci. U. S. A. 2011

Scott et al.
Nat. Rev. Drug Discovery 2016

Kim et al.
Bioconjugate Chem. 2013

Kim et al.
Bioconjugate Chem. 2016

Ozawa et al.
Anal. Chem. 2013

Okamoto et al.
Curr. Opin. Struct. Biol. 2017

Sun et al.
Int. J. Mol. Sci. 2016

Li et al.
Talanta 2013

Machleidt et al.
ACS Chem. Biol. 2015

Similar Publications