title: 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.
authors.affiliation: Laboratory of Molecular Physiology, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland, United States of America. stevevog@mail.nih.gov
authors.forename: Steven S
authors.initials: SS
authors.lastname: Vogel
authors.forename: Tuan A
authors.initials: TA
authors.lastname: Nguyen
authors.forename: B Wieb
authors.initials: BW
authors.lastname: van der Meer
authors.forename: Paul S
authors.initials: PS
authors.lastname: Blank
mesh_terms: D003198:Computer Simulation; D003624:Darkness; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D009010:Monte Carlo Method
chemical_list: D049452:Green Fluorescent Proteins