SPECTROSCOPY, IMAGING, OTHER TECHNIQUES |
Optical lock-in detection of fluorescence resonance energy transfer using synthetic and genetically-encoded optical switches
Shu Mao 1, Richard KP Benninger 2, Yuling Yan 3, Chutima Petchprayoon 1, David K Jackson 1, Christopher J Easley 4, David W Piston 2 and Gerard Marriott 1*
1 University of Wisconsin
2 Vanderbilt University
3 Stanford University
4 Vanderbilt
* To whom correspondence should be addressed. E-mail: marriott{at}physiology.wisc.edu.
Submitted on October 31, 2007
Revised on December 3, 2007
Accepted on 22 January 2008
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Abstract |
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The Förster resonance energy transfer (FRET) technique is widely used for studying protein interactions within live cells. However, the effectiveness and sensitivity of determining FRET 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) that 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 GFP-alkylguaninetransferase (AGT) fusion protein that is labeled with benzylguanine-NitroBIPS. In vivo imaging studies on this 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.
Key Words:
FRET, Lock-in detection, Optical switches, Protein interactions
