Analysis of FRET Signals in the Presence of Free Donors and Acceptors

Abstract

A method for spectral analysis of Förster resonance energy transfer (FRET) signals is presented, taking into consideration both the contributions of unpaired donor and acceptor fluorophores and the influence of incomplete labeling of the interacting partners. It is shown that spectral analysis of intermolecular FRET cannot yield accurate values of the Förster energy transfer efficiency E, unless one of the interactors is in large excess and perfectly labeled. Instead, analysis of donor quenching yields a product of the form Ef(d)p(a), where f(d) is the fraction of donor-type molecules participating in donor-acceptor complexes and p(a) is the labeling probability of the acceptor. Similarly, analysis of sensitized emission yields a product involving Ef(a). The analysis of intramolecular FRET (e.g., of tandem constructs) yields the product Ep(a). We use our method to determine these values for a tandem construct of cyan fluorescent protein and yellow fluorescent protein and compare them with those obtained by standard acceptor photobleaching and fluorescence lifetime measurements. We call the method lux-FRET, since it relies on linear unmixing of spectral components.

Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs

“…-bound YC3.60 sample again indicates a population of YC3.60 molecules, which do not exhibit FRET. Both the heterogeneity of the fluorescence decay of ECFP and the presence of a population of non-interacting donor molecules in the construct severely complicate quantitative analysis of FRET in terms of discrete lifetimes (Millington et al 2007;Borst et al 2008;Wlodarczyk et al 2008).…”

Genetically Encoded Ca 2+ Indicators in Cardiac Myocytes

“…83 f DA , ε D , and ε A can be regarded as constants within a given biosensor, but their actual values do not need to be known to perform the calculation. To determine the apparent FRET efficiency for yellow cameleon, the following formula is used: …”

In Vivo Bacterial Morphogenetic Protein Interactions

“…Because now not only information is obtained on whether the proteins interact but also on the contribution of each protein to the interaction. The contribution of each component can be quantified by 'linear unmixing' of the spectra (Clegg, 1992;Clegg et al, 1992;Murchie et al, 1989;Wlodarczyk et al, 2008). An elaborate unmixing description fitting to our setup can be found in the supplemental information of the corresponding publication and will be explained in less detail (Alexeeva et al, 2010).…”