Measuring Förster Resonance Energy Transfer
one fl uorescence lifetime ( Table 1 ), it was replaced with the improved variants of mCerulean, mCerulean3 [ 9 ], or mTurquoise [ 10 ]. T ese newer variants have reduced photoswitching behavior and improved photosta- bility compared to mCerulean. Most important for FLIM measurements, both of the improved variants have a single component lifetime ( Table 1 ), which simplifi es the lifetime analysis for FRET experiments described below. We fi nd that mTurquoise and mCerulean3 perform equally well for FRET-FLIM studies [ 11 ].
FLIM measurements of the FRET standards . T e fusion protein consisting of mTurquoise coupled directly to mVenus through the fi ve-amino acid (5aa) linker serves as a high FRET effi ciency standard. Because the fl uorescence lifetime can be very sensitive to the local environment of the donor fl uorophore, a similar fusion protein consisting of mTurquoise linked to a mutated mVenus, called Amber, was also made [ 8 ]. T e mutation converts the chromophore tyrosine to a cysteine producing a non-fl uorescent form of Venus that folds correctly, but does not act as a FRET acceptor, providing an accurate measurement of the unquenched donor lifetime. Finally, a fusion protein consisting of mTurquoise linked to mVenus through the 229-amino acid
Figure 5 : The phasor plot analysis of the lifetimes for the FRET standards produced in living cells. (A) The intensity image and phasor lifetime distribution from a cell expressing Turquoise-5aa-Amber (unquenched donor; the calibration bars are 10 μ m), indicating a single exponential lifetime of 3.9 ns ( Table 2). (B) The intensity image and phasor lifetime distribution from a cell expressing Turquoise-TRAF-Venus, indicating an average lifetime of 3.6 ns ( Table 2). (C) The intensity image and phasor lifetime distribution from a cell expressing Turquoise-5aa-Venus demonstrating a multi-component lifetime. The average amplitude-weighted average lifetime was 2.2 ns ( Table 2 ).
tumor necrosis factor receptor associated factor (TRAF) domain (Turquoise-TRAF-Venus) provides a low FRET effi ciency standard [ 12 ]. T e FRET effi ciency ( E FRET ) is determined from the ratio of the unquenched donor lifetime (τ D ; Turquoise-5aa-Amber) to the quenched donor lifetime in the presence of the acceptor (τ DA ; Turquoise-5aa-Venus, Turquoise-TRAF-Venus):
EFRET =− 1 t t
DA D
(Eq. 1)
T e composite phasor plot in Figure 5 compares the unquenched and quenched donor lifetimes for the diff erent FRET standard fusion proteins produced in living cells. T e intensity images obtained from representative cells expressing the diff erent FRET standard fusion proteins are shown in the top panels of Figure 5 , and the average lifetime measurements for each FRET standard are presented in Table 2 . T e average unquenched donor lifetime for Turquoise-5aa-Amber expressed in cells was 3.9 ns ( Table 2 ), and the phasor plot shows it is best fi t to a single-component decay ( Figure 5A ). T e lifetime distribution for the Turquoise-TRAF-Venus fusion protein can be distinguished from that of the
48
unquenched donor ( Figure 5B ) and is shiſt ed to the right along the semicircle indicating a shorter lifetime. T e average donor lifetime for Turquoise-TRAF-Venus expressed in cells was 3.5 ns, which corresponds to an FRET effi ciency of 10%, determined using Eq. 1 ( Table 2 ). In sharp contrast, the lifetime distribution for the cell expressing Turquoise-5aa- Venus shows the quenched donor lifetime for the high FRET standard ( Figure 5C ). T e lifetime distribution for the high FRET standard is shiſt ed far to the right relative to the unquenched donor and falls inside the semicircle, indicating a multi-component lifetime. When analyzing the multi- exponential lifetimes that result from energy transfer, the E FRET is determined using the amplitude-weighted lifetime [ 13 ]. T e average amplitude-weighted lifetime for Turquoise-5aa-Venus was 2.2 ns, corresponding to an E FRET of 43% ( Table 2 ). FLIM measurements of protein-protein interactions . Because FLIM directly measures the quenching of the donor resulting from FRET, and does not require correction for SBT or assumptions in the data analysis, the interpretation of intermolecular FRET measurements of protein-protein interactions is simplified compared to other methods [ 2 – 7 ]. However, because the donor- and acceptor-labeled proteins are produced independently of one another in intermolecular
www.microscopy-today.com • 2015 May
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