Abstract
Most fluorescent proteins exhibit multiexponential fluorescence decays, indicating a heterogeneous excited state population. FRET between fluorescent proteins should therefore involve multiple energy transfer pathways. We recently demonstrated the FRET pathways between EGFP and mCherry (mC), upon the dimerization of 3-phosphoinositide dependent protein kinase 1 (PDK1), to be highly restricted. A mechanism for FRET restriction based on a highly unfavorable κ2 orientation factor arising from differences in donor–acceptor transition dipole moment angles in a far from coplanar and near static interaction geometry was proposed. Here this is tested via FRET to mC arising from the association of glutathione (GSH) and glutathione S-transferase (GST) with an intrinsically homogeneous and more mobile donor Oregon Green 488 (OG). A new analysis of the acceptor window intensity, based on the turnover point of the sensitized fluorescence, is combined with donor window intensity and anisotropy measurements which show that unrestricted FRET to mC takes place. However, a long-lived anisotropy decay component in the donor window reveals a GST-GSH population in which FRET does not occur, explaining previous discrepancies between quantitative FRET measurements of GST-GSH association and their accepted values. This reinforces the importance of the local donor–acceptor environment in mediating energy transfer and the need to perform spectrally resolved intensity and anisotropy decay measurements in the accurate quantification of fluorescent protein FRET.
Highlights
Förster Resonance Energy Transfer (FRET) describes the nonradiative transmission of electronic energy from a donor molecule to a nearby acceptor due to dipole−dipole coupling.[1,2] FRET measurements have found widespread application in the study of nanoscale processes in the biosciences, such as changes in conformation and intermolecular interactions.[3,4] FRET is well understood for homogeneous populations of donors and acceptors,[2,5] but in recent years, the use of genetically encodable fluorescent protein FRET pairs has become widespread.[6]
By combining time-resolved fluorescence intensity and anisotropy measurements of the Oregon Green 48824 (OG)-GSH-glutathione S-transferase (GST)-mC complex, we have shown that the state restriction observed in fluorescent protein to fluorescent protein FRET is relaxed when the donor is replaced by a more mobile synthetic fluorophore
This demonstrates that the restricted geometry of a fluorescent protein tandem construct, which will remain effectively static on the time scales over which FRET occurs, is a significant cause of the differential energy transfer dynamics between the heterogeneous excited state populations.[15]
Summary
Förster Resonance Energy Transfer (FRET) describes the nonradiative transmission of electronic energy from a donor molecule to a nearby acceptor due to dipole−dipole coupling.[1,2] FRET measurements have found widespread application in the study of nanoscale processes in the biosciences, such as changes in conformation and intermolecular interactions.[3,4] FRET is well understood for homogeneous populations of donors and acceptors,[2,5] but in recent years, the use of genetically encodable fluorescent protein FRET pairs has become widespread.[6]. Accurate quantitative application of fluorescent protein FRET is crucially dependent on the correct understanding of the underlying photophysics. This point is strongly evidenced by our recent work on the homodimerization of 3phosphoinositide dependent kinase-1 (PDK1) using the standard FRET pair of enhanced green fluorescent protein (EGFP) and mCherry (mC).[14] Both proteins exhibit intrinsic biexponential fluorescence decays.[15] Combining time-resolved fluorescence intensity and anisotropy measurements of the donor and acceptor, we found that FRET was highly restricted, involving transfer from only one emitting state of EGFP to the minority decay component of mC. When emulating unrestricted FRET by the optical excitation of mC across the donor−acceptor spectral overlap, no such constraint was Received: November 8, 2016 Revised: December 23, 2016 Published: December 29, 2016
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