FRET interactions

FRET interactions are typically characterized by either steady-state or transient fluorescence emission signals from the donor or acceptor species. Efficient nonradiative energy transfer results in donor PL loss associated with acceptor gain in photoluminescence intensity (if the acceptor is an emitter). The rate of this energy transfer is related to the intrinsic lifetime of the isolated donor and depends strongly on the donor-acceptor separation distance ... 

Experimentally, FRET interactions are observed and quantified by measuring the donor or acceptor emission signals. The most common and practical definition of FRET efficiency is... 

A simplified theory of FRET is sufficient to describe affinity sensors used in fluorescence transduction of glucose concentrations. A key quantity that describes the potential FRET interaction between a donor-acceptor pair is the Forster distance, Ro, the distance at which half the donor molecules are quenched by the acceptor molecules. Ro is proportional to several parameters of the fluorophores, in accordance with Ro = K6 Jx2n 4 cf>DJ l], where K is a constant. The variable k2 refers to the relative spatial orientation of the dipoles of D and A, taking on values from 0 to 4 for completely orthogonal dipoles and collinear and parallel transitional dipoles k2 = 4,... 

Equation 2 appears simple because the details of the FRET interaction are contained within the Forster distance (/ o) parameter, Eq. 3, where is the quantum yield of the donor, is the orientation factor, J(k) is the spectral overlap integral, is Avogadro s number, and n is the refractive index of the medium between the donor and acceptor [3]. 

In a recent example, Kim et al. showed how the combination of several types of nanoparticles could also be wisely used for the design of signaling protocols. They designed a FRET-based inhibition assay to determine the avidin concentration in solution with AuNPs and QDs. The ensemble involves the use of streptavidin-conjugated QDs that interact with biotin-AuNPs through well-known streptavidin-biotin chemistry. This system was not luminescent due to FRET interaction between QDs and AuNPs. Addition of avidin to this ensemble caused the luminescence to increase gradually because the AuNPs were displaced from the streptavidin-functionalized QDs as a consequence of avidin-biotin interactions (Fig. 8). 

When a gas comes in contact with a solid surface, under suitable conditions of temperature and pressure, the concentration of the gas (the adsorbate) is always found to be greater near the surface (the adsorbent) than in the bulk of the gas phase. This process is known as adsorption. In all solids, the surface atoms are influenced by unbalanced attractive forces normal to the surface plane adsorption of gas molecules at the interface partially restores the balance of forces. Adsorption is spontaneous and is accompanied by a decrease in the free energy of the system. In the gas phase the adsorbate has three degrees of freedom in the adsorbed phase it has only two. This decrease in entropy means that the adsorption process is always exothermic. Adsorption may be either physical or chemical in nature. In the former, the process is dominated by molecular interaction forces, e.g., van der Waals and dispersion forces. The formation of the physically adsorbed layer is analogous to the condensation of a vapor into a liquid in fret, the heat of adsorption for this process is similar to that of liquefoction. 

Fig. 2 A schematic representation of an HTRF assay for a protein-protein interaction. One protein is tagged with a fluorescent molecule whose emission spectra overlaps with the excitation of another fluorescent molecule. When they are in close proximity (above) the energy is transferred. When they diffuse apart (below) or are inhibited from coming together by a small molecule no FRET occurs...

As targets become increasing more complex, the assays used to measure them become complex as well. This is most evident in the current assays which measure protein-protein interactions. Most involve tagging the interacting partners with a variety of either FRET partners or F-Q pairs, or measuring via FP changes [48]. 

Direct interactions between the reference and reporter dyes leading to PET or FRET in this approach should be avoided. 

Exploration of collective effects in multiple transfers that appear when the donor and acceptor are the same molecules and display the so-called homotransfer. In this case, the presence of only one molecular quencher can quench fluorescence of the whole ensemble of emitters coupled by homotransfer [32]. The other possibility of using homo-FRET is the detection of intermolecular interactions by the decrease of anisotropy [33]. 

Tramier M, Gautier I, Piolot T, Ravalet S, Kemnitz K, Coppey J, Durieux C, Mignotte V, Coppey-Moisan M (2002) Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells. Biophys J 83 3570-3577... 

In order to determine whether compounds identified in the primary HTS screen are specific, a counterscreen is required to identify and eliminate false positives that will arise in the primary screen. For protein—protein interaction screens, it is preferable to test an unrelated protein pair that uses the same mode of detection. For our purposes, we adapted a previously described TR-FRET assay that monitors the interaction between bacterial Staphylococcus aureus Dnal and phage protein 77ORF104 (Liu et al., 2004). 

FRET is a nonradiative process that is, the transfer takes place without the emission or absorption of a photon. And yet, the transition dipoles, which are central to the mechanism by which the ground and excited states are coupled, are conspicuously present in the expression for the rate of transfer. For instance, the fluorescence quantum yield and fluorescence spectrum of the donor and the absorption spectrum of the acceptor are part of the overlap integral in the Forster rate expression, Eq. (1.2). These spectroscopic transitions are usually associated with the emission and absorption of a photon. These dipole matrix elements in the quantum mechanical expression for the rate of FRET are the same matrix elements as found for the interaction of a propagating EM field with the chromophores. However, the origin of the EM perturbation driving the energy transfer and the spectroscopic transitions are quite different. The source of this interaction term... 

Fig. 5.4. Correlation between R0 and FRET efficiency. Exchanging ECFP/ EYFP (R0 = 4.72 nm, dashed line) for the red-shifted VFP FRET-pair mKO/ mCherry (R0 = 6.37 nm, solid line) will increase the measured FRET efficiency, since the distance between donor and acceptor is expected to remain unchanged. A FRET pair with increased R0 yields detectable FRET over longer distances and can be used to measure protein-protein interaction between larger proteins.

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