Optical lock-in detection of FRET using synthetic and genetically encoded optical switches.
The Förster resonance energy transfer (FRET) technique is widely used for studying protein interactions within live cells. The effectiveness and sensitivity of determining FRET, however, can be reduced by photobleaching, cross talk, autofluorescence, and unlabeled, endogenous proteins. We present a FRET imaging method using an optical switch probe, Nitrobenzospiropyran (NitroBIPS), which substantially improves the sensitivity of detection to <1% FRET efficiency. Through orthogonal optical control of the colorful merocyanine and colorless spiro states of the NitroBIPS acceptor, donor fluorescence can be measured both in the absence and presence of FRET in the same FRET pair in the same cell. A SNAP-tag approach is used to generate a green fluorescent protein-alkylguaninetransferase fusion protein (GFP-AGT) that is labeled with benzylguanine-NitroBIPS. In vivo imaging studies on this green fluorescent protein-alkylguaninetransferase (GFP-AGT) (NitroBIPS) complex, employing optical lock-in detection of FRET, allow unambiguous resolution of FRET efficiencies below 1%, equivalent to a few percent of donor-tagged proteins in complexes with acceptor-tagged proteins.
Mao
Shu
S
Department of Physiology, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Benninger
Richard K P
RK
Yan
Yuling
Y
Petchprayoon
Chutima
C
Jackson
David
D
Easley
Christopher J
CJ
Piston
David W
DW
Marriott
Gerard
G
D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism*; D055095:Optics and Photonics; D015202:Protein Engineering / Q000379:methods*; D025941:Protein Interaction Mapping / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism*
D005456:Fluorescent Dyes; D011994:Recombinant Proteins