The Impact of Heterogeneity and Dark Acceptor States on FRET: Implications for Using Fluorescent Protein Donors and Acceptors

Abstract

Förster resonance energy transfer (FRET) microscopy is widely used to study protein interactions in living cells. Typically, spectral variants of the Green Fluorescent Protein (FPs) are incorporated into proteins expressed in cells, and FRET between donor and acceptor FPs is assayed. As appreciable FRET occurs only when donors and acceptors are within 10 nm of each other, the presence of FRET can be indicative of aggregation that may denote association of interacting species. By monitoring the excited-state (fluorescence) decay of the donor in the presence and absence of acceptors, dual-component decay analysis has been used to reveal the fraction of donors that are FRET positive (i.e., in aggregates)._However, control experiments using constructs containing both a donor and an acceptor FP on the same protein repeatedly indicate that a large fraction of these donors are FRET negative, thus rendering the interpretation of dual-component analysis for aggregates between separately donor-containing and acceptor-containing proteins problematic. Using Monte-Carlo simulations and analytical expressions, two possible sources for such anomalous behavior are explored: 1) conformational heterogeneity of the proteins, such that variations in the distance separating donor and acceptor FPs and/or their relative orientations persist on time-scales long in comparison with the excited-state lifetime, and 2) FP dark states.

Quantifying intracellular equilibrium dissociation constants using single‐channel time‐resolved FRET

“…We have demonstrated this methodology with the widely used eGFP -mCherry FRET pair, but other donor-acceptor pairs also report a reduced maximal f RET [2,13,14], suggesting that that our approach for estimating the intracellular K d of intermolecular interactions can be extended to a large range of fluorescent-protein pairs.…”

“…Although orientational heterogeneity as a result of the flexibility of the linkers between the fluorophores and the proteins they are fused to average out any orientational mismatch while adding to the complexity of the decay [13], its effect is expected to be small due to physical restrictions arising from the combination of the short linker length (less than 17 aminoacids, SM) and the size of the fluorophore barrels. This is consistent with previous in-vitro observations of little local motion of eGFP and mCherry when fused to PDK1 [12].…”

“…Higher f max RET and E RET will result in more complexes undergoing FRET for the same K d , which facilitates FRET detection, although it may adversely affect the accuracy of K d determination. Also, besides eGFP and mCherry, many donor-acceptor combinations report a reduced maximal f RET [2,13,14], including those with monoexponentially decaying fluorescent proteins such as mCitrine or mTFP1 [1]. Moreover, the relatively large size of fluorescent proteins precludes the chromophores from getting much closer than their Fö rster distance (commonly in the 5 to 6.5 nm range [32]), often resulting in modest E RET that favours the validity of the approximation to [D T ].…”

Measuring Protein Interactions Using Förster Resonance Energy Transfer and Fluorescence Lifetime Imaging Microscopy

“…Since the fluorescence lifetime can be very sensitive to the local environment of the donor fluorophore, a similar plasmid that encodes the mCerulean3 linked to mutant variant of mVenus, called Amber, was also generated. The mutation converts the chromophore tyrosine to a cysteine producing a non-fluorescent form of Venus that folds correctly, but does not act as a FRET acceptor [24]. Thus, the local environment for Cerulean3 is same for the Amber and Venus constructs, with the Cerulean3-5aa-Amber fusion protein providing an accurate measurement of the unquenched donor lifetime.…”

“…Since there will be a negligible change in the dipole orientation during the excited state lifetime, little averaging will occur. Monte-Carlo simulations were recently used to demonstrate that the slow rotation of the FPs has the potential to give rise to a bimodal distribution of efficiencies in FRET experiments [24]. When analyzing the multi-exponential lifetimes that result from the linked FRET standard protein, the energy transfer efficiency is determined using the amplitude-weighted lifetime [21].…”