Characterization of an improved donor fluorescent protein for Forster resonance energy transfer microscopy.
The genetically encoded fluorescent proteins (FP), used in combination with Forster resonance energy transfer (FRET) microscopy, provide the tools necessary for the direct visualization of protein interactions inside living cells. Typically, the Cerulean and Venus variants of the cyan and yellow FPs are used for FRET studies, but there are limitations to their use. Here, Cerulean and the newly developed monomeric Teal FP (mTFP) are compared as FRET donors for Venus using spectral and fluorescence lifetime measurements from living cells. The results demonstrate that when compared to Cerulean, mTFP has increased brightness, optimal excitation using the standard 458-nm laser line, increased photostability, and improved spectral overlap with Venus. In addition, the two-photon excitation and fluorescence lifetime characteristics are determined for mTFP. Together, these measurements indicate that mTFP is an excellent donor fluorophore for FRET studies, and that its use may improve the detection of interactions involving proteins that are difficult to express, or that need to be produced at low levels in cells.
Day
Richard N
RN
University of Virginia Health System, Departments of Medicine and Cell Biology, P. O. Box 800578, Charlottesville, Virginia 22908-0578, USA. rnd2v@virginia.edu
Booker
Cynthia F
CF
Periasamy
Ammasi
A
D000818:Animals; D002478:Cells, Cultured; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D007089:Image Enhancement / Q000379:methods; D008164:Luminescent Proteins / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D010902:Pituitary Gland / Q000378:metabolism*; D025941:Protein Interaction Mapping / Q000379:methods*; D012680:Sensitivity and Specificity
D008164:Luminescent Proteins