title: Time-resolved homo-FRET studies of biotin-streptavidin complexes.
abstract: Förster resonance energy transfer is a mechanism of fluorescence quenching that is notably useful for characterizing properties of biomolecules and/or their interactions. Here we study water-solutions of Biotin-Streptavidin complexes, in which Biotin is labeled with a rigidly-bound fluorophore that can interact by Förster resonance energy transfer with the fluorophores labeling the other, up to three, Biotins of the same complex. The fluorophore, Atto550, is a Rhodamine analogue. We detect the time-resolved fluorescence decay of the fluorophores with an apparatus endowed with single-photon sensitivity and temporal resolution of ~30ps. The decay profiles we observe for samples containing constant Biotin-Atto550 conjugates and varying Streptavidin concentrations are multi-exponential. Each decay component can be associated with the rate of quenching exerted on each donor by each of the acceptors that label the other Biotin molecules, depending on the binding site they occupy. The main features that lead to this result are that (i) the transition dipole moments of the up-to-four Atto550 fluorophores that label the complexes are fixed as to both relative positions and mutual orientations; (ii) the fluorophores are identical and the role of donor in each Biotin-Streptavidin complex is randomly attributed to the one that has absorbed the excitation light (homo-FRET). Obviously the high-temporal resolution of the excitation-detection apparatus is necessary to discriminate among the fluorescence decay components.
authors: Department of Science and High Technology, University of Insubria, Como 22100, Italy. Electronic address: Alessandra.Andreoni@uninsubria.it.
authors: Alessandra
authors: A
authors: Andreoni
authors: Department of Science and High Technology, University of Insubria, Como 22100, Italy. Electronic address: Luca.Nardo@uninsubria.it.
authors: Luca
authors: L
authors: Nardo
authors: Department of Medical Biophysics, Karolinska Institutet, 17 177 Stockholm, Sweden; Laboratory of Biomedical Optics, Swiss Federal Institute of Technology, 1015 Lausanne, Switzerland. Electronic address: rudolf.rigler@bluewin.ch.
authors: Rudolf
authors: R
authors: Rigler
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D001710:Biotin / Q000031:analogs & derivatives* / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D007700:Kinetics
chemical_list: D001426:Bacterial Proteins; D005456:Fluorescent Dyes; C066114:biotin-streptavidin complex; D001710:Biotin
keywords: Biotin-streptavidin complexes; Fluorescence decay; Förster resonance energy transfer; Homo-FRET; Picosecond resolution