title: Restricted state selection in fluorescent protein Förster resonance energy transfer.
abstract: The measurement of donor lifetime modification by Förster resonance energy transfer (FRET) is a widely used tool for detecting protein-protein interactions and protein conformation change. Such measurements can be compromised by the presence of a significant noninteracting fraction of molecules. Combining time-resolved intensity and anisotropy measurements gives access to both molecular distance and orientation. Fluorescent proteins frequently used to detect energy transfer in biological systems often exhibit decay characteristics indicative of more than one excited state. However, little attention has thus far been given to the specific modes of energy transfer, in particular, which states are predominantly coupled. Here, we use a previously characterized dimerization system to study energy transfer between EGFP and mCherry. Optically excited EGFP and mCherry both exhibit biexponential decays, and FRET should therefore involve dipole-dipole transfer between these four states. Analysis of the sensitized fluorescence anisotropy and intensity decays indicates that FRET transfer is predominantly from the shorter lived EGFP emitting state (2.43 ns) to the longer lived (ca. 2.77 ns) minority component (ca. 16%) of the optically excited mCherry emission. This high degree of state selection between these two widely used FRET pairs highlights the fundamental differences that can arise between direct optical excitation of an isotropic molecular population and dipole-dipole coupling in a far from isotropic interaction geometry and has consequences regarding the accurate interpretation of fluorescent protein FRET data.
authors.affiliation: Department of Physics and Astronomy, University College London, Gower Street, London WC1E 6BT, United Kingdom.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Masters
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Marsh
authors.forename: Daven A
authors.initials: DA
authors.lastname: Armoogum
authors.forename: Nick
authors.initials: N
authors.lastname: Nicolaou
authors.forename: Banafshé
authors.initials: B
authors.lastname: Larijani
authors.forename: Angus J
authors.initials: AJ
authors.lastname: Bain
mesh_terms: D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry*; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase
chemical_list: D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D017346:Protein Serine-Threonine Kinases