Fluorescent Protein Based FRET Pairs with Improved Dynamic Range for Fluorescence Lifetime Measurements

<div><p>Fluorescence Resonance Energy Transfer (FRET) using fluorescent protein variants is widely used to study biochemical processes in living cells. FRET detection by fluorescence lifetime measurements is the most direct and robust method to measure FRET. The traditional cyan-yellow fluorescent protein based FRET pairs are getting replaced by green-red fluorescent protein variants. The green-red pair enables excitation at a longer wavelength which reduces cellular autofluorescence and phototoxicity while monitoring FRET. Despite the advances in FRET based sensors, the low FRET efficiency and dynamic range still complicates their use in cell biology and high throughput screening. In this paper, we utilized the higher lifetime of NowGFP and screened red fluorescent protein variants to develop FRET pairs with high dynamic range and FRET efficiency. The FRET variations were analyzed by proteolytic activity and detected by steady-state and time-resolved measurements. Based on the results, NowGFP-tdTomato and NowGFP-mRuby2 have shown high potentials as FRET pairs with large fluorescence lifetime dynamic range. The <i>in vitro</i> measurements revealed that the NowGFP-tdTomato has the highest Förster radius for any fluorescent protein based FRET pairs yet used in biological studies. The developed FRET pairs will be useful for designing FRET based sensors and studies employing Fluorescence Lifetime Imaging Microscopy (FLIM).</p></div

Photobleaching of NowGFP and EGFP in <i>E</i>. <i>coli</i> cells.

<p>Bacterial cells expressing the fluorescent proteins were excited at 488 nm laser, and the photobleaching was analyzed from images acquired using confocal microscope equipped with 63x oil objective. The difference in photobleaching of NowGFP and EGFP at different laser intensities (141 μW and 23 μW) can be observed from the figure. </p

Variations in the FRET response of FRET pairs on proteolytic cleavage over time.

<p>The control is NowGFP-mRuby2 FRET pair without thrombin cleavage site (LVPS instead of LVPR). ΔR/R was computed as (R₀-R_F)/R₀ where R is donor:acceptor ratio and R₀ is the donor:acceptor ratio when there is no FRET and R_F is the FRET ratio</p

SDS PAGE displaying the proteolytic activity.

<p>The absence of fusion protein band and the presence of the cleaved protein band is visible from Lane 2 and 4 confirming proteolytic cleavage. The dotted arrows indicate the cleaved product and the straight arrow indicate the fusion protein band. </p

Emission spectra of the FRET constructs.

<p>Fluorescence emission spectrum (at 480 nm excitation) of the FRET constructs treated with thrombin. The schematics of the FRET constructs are displayed above the spectrum. "LVPR" represents the sequence GGGSLVPRGS. The decrease in the FRET in time as a result of proteolytic cleavage can be observed from the spectrum. </p

Spectral properties of the fluorescent proteins along with the Förster radius of the FRET pairs.

<p>* With NowGFP as donor (This study)</p><p>† Values from ref. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134436#pone.0134436.ref033" target="_blank">33</a>]</p><p>‡ Values from ref. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134436#pone.0134436.ref002" target="_blank">2</a>]</p><p>§ Values from ref. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134436#pone.0134436.ref046" target="_blank">46</a>]</p><p>Spectral properties of the fluorescent proteins along with the Förster radius of the FRET pairs.</p

Fluorescence lifetime and FRET efficiency of the FRET pairs inside bacterial cells (From an average of approximately 35 individual cells, for each fluorescent protein/pair).

<p>E—FRET efficiency (calculated according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134436#pone.0134436.e003" target="_blank">Eq 3</a>). χ2—calculated standard weighted least squares to assess the goodness of the fit.</p

Intracellular FLIM of <i>E</i>. <i>coli</i> cells.

<p><i>(A)</i> Fluorescence lifetime image of the cells displaying FRET. The cells are excited at 483 nm and the selective emission from the donor was monitored through band pass filter (510/20 nm). NowGFP is the cells expressing donor alone and the variation in lifetime as a result of FRET can be observed from the cells expressing the FRET pairs. The average lifetime is calculated from approximately 30 cells. Image size is 10 μm × 10 μm. <i>(B)</i> Fluorescence decay curve from the cells showing FRET. The decrease in the fluorescence lifetime due to FRET can be observed from the decay curve.</p

Fluorescence lifetime and FRET efficiency of the FRET pairs <i>in vitro</i>.

<p>* Average from all the FRET pairs after thrombin treatment. E—FRET efficiency (calculated according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134436#pone.0134436.e003" target="_blank">Eq 3</a>). χ2—calculated standard weighted least squares to assess the goodness of the fit</p><p>Fluorescence lifetime and FRET efficiency of the FRET pairs <i>in vitro</i>.</p

Stereoview of the nearest chromophore environment of mKillerOrange.

<p><i>Trans-cis</i> (~85%) and <i>cis-cis</i> (~15%) conformations of the chromophore are shown in orange and yellow, respectively. (A) A complete set of residues surrounding the chromophore. (B) Residues forming H-bond network around the chromophore.</p