13 July 2007 Analyzing the influence of contact-induced quenching processes on Förster resonance energy transfer
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Experiments based on Förster resonance energy transfer (FRET) are widely used to obtain information on conformational dynamics of biomolecular systems. To reliably measure FRET, accurate knowledge of photophysical properties of the used fluorophores is indispensable. In high FRET constructs donor (D) and acceptor (A) fluorophores can approach each other close enough that electronic interactions might occur. When separated by distances on the order of van der Waals radii, photophysical properties can be changed reversibly, opening new non-radiative relaxation pathways, or irreversibly, chemically altering the fluorophores. Even transient contacts can thus compromise accurate FRET measurements. To study FRET and competing D-A contact-induced processes we labeled the amino acid cystein (Cys) with two fluorophores. A donor (D; TMR or Cy3B) was attached to the thiol group and an acceptor (A; Atto647N) to the amino group of Cys. Absorption spectroscopy, steady-state fluorescence spectroscopy, and time-correlated single-photon counting (TCSPC) were used to characterize the different A-Cys-D complexes at the ensemble level. In addition, we performed single-molecule FRET experiments using alternating-laser excitation to study the heterogeneity of the FRET-systems. We identified competing quenching processes severely changing D and A quantum yields upon fluorophore contact. The results are applicable for quantitative analysis of FRET in dynamic molecular systems that allow transient contact between D and A fluorophores.
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Ralf Brune, Ralf Brune, Sören Doose, Sören Doose, Markus Sauer, Markus Sauer, } "Analyzing the influence of contact-induced quenching processes on Förster resonance energy transfer", Proc. SPIE 6633, Biophotonics 2007: Optics in Life Science, 66331M (13 July 2007); doi: 10.1117/12.727853; https://doi.org/10.1117/12.727853

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