title: FRET spectrometry: a new tool for the determination of protein quaternary structure in living cells.
abstract: Förster resonance energy transfer (FRET) is an exquisitely sensitive method for detection of molecular interactions and conformational changes in living cells. The recent advent of fluorescence imaging technology with single-molecule (or molecular-complex) sensitivity, together with refinements in the kinetic theory of FRET, provide the necessary tool kits for determining the stoichiometry and relative disposition of the protomers within protein complexes (i.e., quaternary structure) of membrane receptors and transporters in living cells. In contrast to standard average-based methods, this method relies on the analysis of distributions of apparent FRET efficiencies, E(app), across the image pixels of individual cells expressing proteins of interest. The most probable quaternary structure of the complex is identified from the number of peaks in the E(app) distribution and their dependence on a single parameter, termed pairwise FRET efficiency. Such peaks collectively create a unique FRET spectrum corresponding to each oligomeric configuration of the protein. Therefore, FRET could quite literally become a spectrometric method--akin to that of mass spectrometry--for sorting protein complexes according to their size and shape.
authors.affiliation: Physics Department, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin; Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin. Electronic address: vraicu@uwm.edu.
authors.forename: Valerică
authors.initials: V
authors.lastname: Raicu
authors.forename: Deo R
authors.initials: DR
authors.lastname: Singh
mesh_terms: D002470:Cell Survival; D031541:Fluorescence Resonance Energy Transfer; D020836:Protein Structure, Quaternary; D011506:Proteins / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000166:cytology
chemical_list: D011506:Proteins