About the common features behind the mechanism of molecular rotors, photoswitching of engineered fluorescent proteins (GFPs), and Förster resonance energy transfer (FRET). Besides explining the common mechanism, we also offer a new method for increasing sensitivity of viscosity determination even in living cells, by the combination of polarization energy transfer (polFRET) method and a molecular rotor dye either as an energy donor or acceptor.
Keywords: Internal Charge Transfer (TICT) State; Viscosity; Fluorescence Lifetime; Quantum Yield; Photoactivation; Polarization Energy Transfer (polFRET)
Abbreviations: FRET: Förster Resonance Energy Transfer; FRAP: Fluorescence Recovery After Photobleaching; LE: Locally Excited State
Viscosity represents the strength of interaction of an object or a molecule with other objects or molecules within immediate vicinity in a solution. Its importance follows from the fact that via its involvement in the lateral and rotational diffusion coefficients it determines the rates of biochemical-biological processes in almost every area in life sciences. From another point of view, it determines also the rates of energy dissipation of moving bodies, molecules. An interesting feature is that while lateral diffusion depends on the inverse 1st power of the object radius, the rotational one depends on the inverse 3rd power of the radius, i.e. on the volume , suggesting a rule – which is a rather general one, having significance also in fluorescence – that by increasing dimensionality of motion, the degrees of freedom for energy dissipation is also increased, exemplified by translation and rotation as 1- and 3-D motions (Figure 1, Panel A).
Tradional approaches of viscosity measurements in cell biology rest on Fluorescence Recovery After Photobleaching (FRAP) mea surements of translational diffusion and fluorescence or phosphorescence anisotropy measurements of rotational diffusion [2-6]. In FRAP, lateral diffusion is quantified by the speed of reappearance of fluorescence in a target volume after selectively destroying by intense light a portion of fluorophores initially present there [2,3]. In fluorescence anisotropy measurements, rotational diffusion is quantified by the speed of randomization of polarization directions of the emitted photons after excitation by linearly polarized light [4-6]. These two approaches, and even their combinations in non-imaging and imaging modes, could be realized equally well in the cytoplasm, on the cell membrane, and in the different intracellular organelles and/or in their membranes [2,6]. Substantial interest has been arisen by a class of special dyes called molecular rotors  (Figure 1, Panel B).
These are small compounds having several aromatic rings with substantial rotational freedoms around one or more bonds. They have two excited states: In the Locally Excited One (LE) all their rings are in a single plane leading to high quantum efficiency because of the suppression of rotations. In the other, called “Twisted Internal Charge Transfer” (TICT) state, the molecule assumes a 3-D state instead of the original 2-D one, with one or more rings being rotated out from the LE plane [1,7]. Because the dipole moment of the dye is also increased here, this state has also a higher reactivity with the neighborhood, implying also a more efficient loss of energy and a lower quantum efficiency. The point in this process is that the transition from the 2-D planar state to the 3-D non-planar one is basically dictated by the local viscosity and polarity in the immediate vicinity of the dye, with relative sensitivities to these factors controlled by chemical engineering of side groups. As an extreme case, molecular rotors sensitive to only viscosity can be achieved.
This process can also be envisioned as a special type photo- switching between the high quantum efficiency planar state and the lower quantum efficiency TICT state (Figure 1, Panel C). The transition to the TICT state may also be followed by a red-shift in the color of emission for TICT state energy gaps slightly lower than in the LE state. Furthermore, fluorescence emission might also completely cease for much lower TICT state energy gaps. Experimentally, molecular rotors can quantify an increasing local viscosity by increasing fluorescence lifetime, quantum efficiency, fluorescence intensity, and a blue-shifted fluorescence color [7,8]. The outstanding feature of molecular rotors distinguishing them from polarization- based sensing of viscosity, is the much higher sensitivity, due to the fact that only the rotation of a subportion of a dye occurring in ~psec time is involved, i.e. due to the much smaller inertia . Rotor dyes can be engineered not only for sensing viscosity, but also for indicating contact points between objects, and probing binding reactions .
In their work, H Ying et al.  developed a rotor dye by a series of chemical modification of BODIPY for serving two purposes at the same time: The 1st purpose is to enable it for adhering to the vicinal dithiol moieties of endoplasmatic reticulum nascent proteins – via an introduction of an arsenicate moiety – and triggering autophagy of endoplamic reticulum (“reticulophagy”) by this binding. The 2nd purpose is the quantitation of this binding via the above “rotor effects”. The authors have chosen fluorescence lifetime as the probe parameter, to exclude possible disturbances due to changes in local dye concentrations inherent in intensity measurements. An advantage of the rotor dye is that it is adequate to apply a single emission color channel for quantitating viscosity. The other detection channels might be preserved for other dyes for the localization of rotor dye accumulation e.g. lysotracker, mitotracker for lysosomes and mitochondria, respectively. In the above we saw that excitation induced segmental rotation of certain dyes damped by local viscosity may act as a kind of a photoswitching process.
Actually, the operation of the “official” photoswitching or photoactivation phenomenon taking place in certain engineered Visible Fluorescent Proteins (VFPs) rests on a similar principle [11,12]. There the chomophore suspended in a b-barrel shield might have large enough rotational freedom to sample different orientations after excitation. The photo-activation property is conveyed by the circumstance that these sampled orientations might have different quantum yields due to the different degree of quenching by the amino acid side chains of the b-barrel wall. The energy for the rotation of the choromophore is supplied by the excitation: The increased dipole moment of the chromophore upon excitation results in increased torque exerted by the neighboring amino acid side chains of the b-barrel wall. Photoactivation might be tuned by the careful engineering of chromophore as well as the quencher amino acid side chains down the barrel wall. The conventional Förster Resonance Energy Transfer (FRET) pair has already the main characteristics of a rotor dye, notwithstanding now that the relative orientation of two different dyes is that what is important (Figure 1, Panel D).
The conventional Förster responsible for switching off the donor – and switching on the acceptor – at a given donor-acceptor distance is the relative orientation of the absorption dipole of acceptor and the donor local field at the position of acceptor [13,14]. The functional form of this dependence is conveyed by orientation factor for FRET (k2). At each position around the donor it is generally valid that k2 is maximal when the acceptor dipole is parallel, and zero when it is perpendicular with the direction of donor local field at that special position. It also can be suspected that by increasing the speed of rotation of either of the donor or acceptor might lead to increased FRET, by the increased frequency of parallel encounters [15,16] (Figure 2). To put it another way, k2 and FRET efficiency might be increased by reducing local viscosity [15,17]. FRET has already been combined with the rotor dyes for sensing local viscosity [7,18]. The problem of sensing viscosity via rotor intensity is that intensity depends not only on viscosity but also on the concentration of the rotor dye.
In the “ratiometric” arrangement, a rotor dye and a conventional one are coupled together with a flexible linker realizing a FRET-pair of the 1-to-1 donor-acceptor ratio. Here the fluorescence emission of the conventional dye – supposed to be insensitive to viscosity – is used for probing local concentration of FRET-pair and rotor dye, and the fluorescence of the rotor for quantitating viscosity. Recently “polarization energy transfer” (polFRET) methods has been introduced to monitor donor and acceptor dye rotations at different distances [19,20] (Figure 3). Here, polarized donor and acceptor intensity components are measured simultaneously, enabling quantitation of donor and acceptor anisotropies beside FRET efficiency. Although this method as it stands might already be useful for sensing viscosity, its capabilities might be improved by the involvement of rotor dyes:
(i) Rotor dye response might be calibrated in the above-described “ratiometric” fashion, when the intensity of conventional dye is used for concentration indicator. A direct measurement of also the rotational correlation time of the conventional dye, could serve as another check of viscosity in addition to the rotor intensity.
(ii) Sensitivity on conformational changes and binding reactions of the polFRET method might be improved with rotor dyes. If e.g. rotor is applied as donor and conventional dye as acceptor, increase in viscosity would introduce increase in donor and acceptor anisotropies, and quantum efficiency of the donor. Even if donor-acceptor distance is left unaltered, and k2 might diminish with increasing viscosity, an increase in FRET efficiency could still be anticipated based on the dependence of FRET rate constant on the donor quantum yield.
If, however, by reversing the above scheme, rotor would be applied as acceptor and conventional dye as donor, donor and acceptor anisotropies and acceptor emission intensity would increase with viscosity. At a fixed donor-acceptor separation, FRET efficiency would now only decrease due to the decrease of k2 with increasing viscosity. Donor and acceptor anisotropy enhancements would serve as a check for the intensity enhancement of the rotor with increasing viscosity, valid also for the previous case.
We revealed parallel features behind the working meahanisms of molecular rotors, photoswitching of GFPs, and the FRET process. Polarisation FRET (polFRET) method is suggested in combination with a molecular rotor either as a donor or an acceptor for increasing the sensitivity of viscosity measurements in a fluorescent microscope or in a flow cytometer.
Financial support to L.B. for this work was provided by TÁMOP- 4.2.2.A-11/1/KONV-2012-0045 project co-financed by the European Union and the European Social Fund, OTKA Bridging Fund support OSTRAT/810/213 by the University of Debrecen. L.B. is wishing to express the appreciation for Imre Péntek (Nagykőrös, Hungary) for a fascinating first introduction into the field of wave polarization.
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