title: Experimental verification of the kinetic theory of FRET using optical microspectroscopy and obligate oligomers.
abstract: Förster resonance energy transfer (FRET) is a nonradiative process for the transfer of energy from an optically excited donor molecule (D) to an acceptor molecule (A) in the ground state. The underlying theory predicting the dependence of the FRET efficiency on the sixth power of the distance between D and A has stood the test of time. In contrast, a comprehensive kinetic-based theory developed recently for FRET efficiencies among multiple donors and acceptors in multimeric arrays has waited for further testing. That theory has been tested in the work described in this article using linked fluorescent proteins located in the cytoplasm and at the plasma membrane of living cells. The cytoplasmic constructs were fused combinations of Cerulean as donor (D), Venus as acceptor (A), and a photo-insensitive molecule (Amber) as a nonfluorescent (N) place holder: namely, NDAN, NDNA, and ADNN duplexes, and the fully fluorescent quadruplex ADAA. The membrane-bound constructs were fused combinations of GFP2 as donor (D) and eYFP as acceptor (A): namely, two fluorescent duplexes (i.e., DA and AD) and a fluorescent triplex (ADA). According to the theory, the FRET efficiency of a multiplex such as ADAA or ADA can be predicted from that of analogs containing a single acceptor (e.g., NDAN, NDNA, and ADNN, or DA and AD, respectively). Relatively small but statistically significant differences were observed between the measured and predicted FRET efficiencies of the two multiplexes. While elucidation of the cause of this mismatch could be a worthy endeavor, the discrepancy does not appear to question the theoretical underpinnings of a large family of FRET-based methods for determining the stoichiometry and quaternary structure of complexes of macromolecules in living cells.
authors: Physics Department, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin.
authors: Suparna
authors: S
authors: Patowary
authors: Ontario Brain Institute, Ontario, Toronto, Canada.
authors: Luca F
authors: LF
authors: Pisterzi
authors: Physics Department, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin.
authors: Gabriel
authors: G
authors: Biener
authors: Physics Department, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin.
authors: Jessica D
authors: JD
authors: Holz
authors: Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin.
authors: Julie A
authors: JA
authors: Oliver
authors: The Leslie L. Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada.
authors: James W
authors: JW
authors: Wells
authors: Physics Department, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin; Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin. Electronic address: vraicu@uwm.edu.
authors: Valerică
authors: V
authors: Raicu
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins