Quantification of Förster resonance energy transfer by monitoring sensitized emission in living plant cells

Abstract

Förster resonance energy transfer (FRET) describes excitation energy exchange between two adjacent molecules typically in distances ranging from 2 to 10 nm. The process depends on dipole-dipole coupling of the molecules and its probability of occurrence cannot be proven directly. Mostly, fluorescence is employed for quantification as it represents a concurring process of relaxation of the excited singlet state S1 so that the probability of fluorescence decreases as the probability of FRET increases. This reflects closer proximity of the molecules or an orientation of donor and acceptor transition dipoles that facilitates FRET. Monitoring sensitized emission by 3-Filter-FRET allows for fast image acquisition and is suitable for quantifying FRET in dynamic systems such as living cells. In recent years, several calibration protocols were established to overcome to previous difficulties in measuring FRET-efficiencies. Thus, we can now obtain by 3-filter FRET FRET-efficiencies that are comparable to results from sophisticated fluorescence lifetime measurements. With the discovery of fluorescent proteins and their improvement toward spectral variants and usability in plant cells, the tool box for in vivo FRET-analyses in plant cells was provided and FRET became applicable for the in vivo detection of protein-protein interactions and for monitoring conformational dynamics. The latter opened the door toward a multitude of FRET-sensors such as the widely applied Ca2+-sensor Cameleon. Recently, FRET-couples of two fluorescent proteins were supplemented by additional fluorescent proteins toward FRET-cascades in order to monitor more complex arrangements. Novel FRET-couples involving switchable fluorescent proteins promise to increase the utility of FRET through combination with photoactivation-based super-resolution microscopy.

Binary 2in1 Vectors Improve in Planta (Co)localization and Dynamic Protein Interaction Studies

“…In addition, the chromophores chosen in this vector set are all monomeric, thereby avoiding dimerization or quenching due to fluorophore interaction (Zacharias et al, 2002;Zhang et al, 2002). While the FRET pairs mEGFP-mCherry (Tramier et al, 2006) and mTRQ2-mVenus (Goedhart et al, 2012) were demonstrated before (summarized by Müller et al, 2013), to our knowledge, the pair mVenus-tagRFP has not been used previously.…”

Resonance energy transfer and competing processes in donor–acceptor of sodium zinc (II)-2,9,16,23-phthalocyanine tetracarboxylate molecule

“…where f d is the fluorescence quantum yield of donor in the absence of the acceptor (quencher), n is the index of refraction of the solvent (n = 1.33 for water), and κ 2 is the orientation factor describing the relative transition dipole moments of the donor and acceptor ranging between −2 and 2 [23]. Its average value is given by 2/3 in a solution when the donor and acceptor rotate rapidly during the lifetime of the excited donor, and therefore the polarization is 400 600 800…”

Techniques for the analysis of protein-protein interactions in vivo

“…5B, green), where emission of the donor leaks into the emission spectrum of the acceptor. To distinguish FRET from bleed-through artifacts using sensitized emission, great care must be taken when choosing the controls, including donor only, acceptor only, and donor and acceptor together, as well as the analysis of spectral data (Müller et al, 2013). A fluorescent protein (here depicted as the cyan-colored crystal structure of a CFP attached to a POI in gray) that absorbs light (blue wavy arrow) causes an electron to be raised to an excited singlet state, S 1 * (blue solid arrow).…”

“…(Gadella et al, 1999). A detailed discussion of individual point mutations, resulting spectral changes, and their implications for FRET can be found elsewhere (Kremers and Goedhart, 2009;Müller et al, 2013).…”