As FRET is strongly dependent on the distance between the two interacting partners, it allows molecules to be studied below the optical resolution limit. Two interaction partners of interest with the appropriate fluorescent labels can be linked together to observe molecular interactions. Genetically encoded proteins and biosensors have been developed for real time imaging of protein interactions or conformational changes of molecules. A biosensor is a sensitive biological element (enzymes or antibodies, for instance) that interacts (binds with or recognizes) the target under study. The main advantage of fluorescent probes is that they do not require cell-invasive procedures to be studied and can be targeted to different locations in the cell [1, 2]. Over 100 different genetically encoded biosensors have been developed for targets like enzymes, ions, oxidation reduction events and other molecular activities. Most of these sensors are FRET-based and fluorescent, thus they are optimal tools for studying molecular events detected in a microscope .
FRET-based sensors normally rely on one or more fluorescent proteins. As a general principle, these sensors convert a molecular event into a change in fluorescence.
There are three main groups of FRET-based biosensors :
- Usually, the binding of the substrate or sensing molecule reveals a conformational change that increases the FRET efficiency between CFP and YFP. Examples: cameleon Ca2+ -sensor as well as sensors for sugars, glutamate, Zn2+, cAMP, cGMP, NO and membrane potential.
- FRET-based sensors working by enzyme activation; upon phosphorylation of the substrate domain by protein kinase, the phosphorylation recognition domain binds to the phosphorylated substrate domain. This in turn causes a change in FRET between CFP and YFP. Examples: kinases and GTPase activity.
- Finally, there are protease-activated FRET-based biosensors. Upon enzyme cleavage the activated protein cleaves a certain sequence that leads to a reduction of FRET between the two partners as the partners become separated from each other. Examples: caspases and matrix metalloproteases.
In the experiment described here, we used a FRET-based protease sensor with a classical design. It contains a CFP and a YFP variant (Cerulean and Citrine) connected by a flexible linker with a random-coiled distribution. An example of a FRET-based protease sensor with enhanced sensitivity is reported by Vinkenborg et al. 2007 . A conformational change within the construct will lead to a change of FRET efficiency between donor and acceptor fluorescence emission. This reaction is independent of the sensor concentration, which is the strength of the FRET-based sensors. When these proteins are incorporated into a protease sensor the emission ratio donor/acceptor rises and FRET can be measured. Upon cleavage at the protease sensor site FRET will no longer occur.
In particular, upon cleavage of a flexible peptide that links Cerulean and Citrine no FRET can be measured any more. This indicates that the peptide linker was cleaved by the protease (see Figure 1).
Fig. 1: Cerulean and Citrine, variants of the fluorescent proteins CFP and YFP, were incorporated into a protease sensor resulting in a rise of the donor/acceptor emission ratio which enabled FRET to be measured. Upon cleavage at the protease sensor site FRET no longer occurs, indicating that the peptide linker was cleaved by the protease.
The change in FRET efficiency can be measured by intensity-based methods (FRET-AB, FRET-Acceptor Bleaching and FRET-SE, FRET-Sensitized Emission). Alternatively, FRET efficiency can be measured based on fluorescence lifetime microscopy (FLIM). FLIM-FRET allows analysis of molecular interactions both in vitro and in vivo.
Here we have used FLIM in the time domain (TCSPC, Time Correlated Single Photon Counting)  to measure FRET in vitro in a biochemical assay using a Cerulean-Citrine construct.
The purified protein construct consisting of two fluorescent proteins Cerulean and Citrine (diluted in buffer) connected only by a flexible peptide linker were studied. Usually, FRET occurs between these two proteins (Figure 1). The flexible peptide linker contains a protease cleavage site in the middle. By cleaving the peptide linker FRET no longer occurs between the fluorescent proteins.
Measurement and results
For FLIM-FRET experiments in general, only the donor molecule is measured. The fluorescence lifetime of Cerulean (donor) in the presence of Citrine (acceptor) was measured using a 440 nm pulsed laser for excitation and a detection range of 456–500 nm. For FLIM measurements in solutions, a low scanning format like 64 x 64 is sufficient as no structure needs to be resolved. Thus, the measurements can be performed faster and more photons can be collected per pixel.
For analysis the average lifetime was computed in the fast FLIM mode using SymPhoTime Software (PicoQuant, Berlin). The lifetime distribution of all image pixels reveals an average fluorescence lifetime t of Cerulean (donor) in the presence of the acceptor, in the (FRET conformation) of 2.5 ns. Then protease K was added to the solution. The fluorescence lifetime of Cerulean was measured again using the same measurement conditions. Now, the average fluorescence lifetime of Cerulean has increased to 3 ns and is therefore significantly longer than before the protein cleavage. This value is in the range of a typical fluorescence lifetime value for unbound (non-FRET) Cerulean. The increase of the donor lifetime indicates that the protein linker was cleaved by the protease and FRET no longer occurs.
As a result we measured a clear shift of 0.5 ns in the average fluorescence lifetime of Cerulean (donor only) in the presence of the acceptor compared to the lifetime of Cerulean after protease cleavage. The FRET efficiency is calculated according to (1 – (t DA/ t D)) x 100. This reveals a FRET efficiency of E = 20 %.
This shift in the lifetime upon protease cleavage is also reflected in the resulting color-coded FLIM images (Figure 2b): Cerulean in the presence of the acceptor reveals a blue FLIM image; Cerulean after protease cleavage shows a yellow-orange FLIM image when using the same color coding scale. The fluorescence lifetime distribution in the images shows a clear shortening of the lifetime upon protease cleavage.
In vitro FLIM-FRET assays are versatile tools for studying different protein constructs regarding their FRET capabilities and FRET efficiencies, allowing an inside view of the molecular processes and conformational changes within constructs. The measurement of FRET efficiency by means of FLIM is a very sensitive method for FRET and has some advantages over intensity-based FRET methods. The excitation of 440 nm is perfect for CFP and its variants like Cerulean. FLIM-FRET measurements of different constructs, sensors, or linkers can be a suitable tool to demonstrate the sensitivity of the complete FRET-based sensor system. Thus, a FRET-based sensor construct can be proven before being used for incorporation into live cells.
The introductory image shows a FLIM recording of isolated thylakoid membranes from Arabidopsis: The spots with the longer lifetime are most likely stacked membrane regions called grana thylakoids. The image is the result of a pixel by pixel fitting with two exponentials using PicoQuant software (SymphoTime). Calculated data are displayed in an RGB false-color model. The amplitudes for the first and second lifetime component are depicted in two different colors. Sample: Courtesy of Dr. Helmut Kirchhoff, Washington State University, Dep. of Plant Molecular Biology, USA.
- Palmer AE, Quin Y, Genevieve J and McCombs JE: Design and application of genetically encoded biosensors. Trends in Biotechnology 29:3 (2011) 144–152.
- Vinkenborg JL, Evers TH, Reulen SWA, Meijer EW Merkx M: Enhanced Sensitivity of FRET-Based Protease Sensors by Redesign of the GFP Dimerization Interface. ChemBioChem 8 (2007) 1119–1121.
- Lakovicz JR: Principles of Fluorescence Spectroscopy, 3rd ed. 103 ff. (2006), Springer New York.