Analyzing the influence of contact-induced quenching processes on Förster resonance energy transfer

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.

[1]  Fred Russell Kramer,et al.  Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes. , 2002, Nucleic acids research.

[2]  Paul R. Selvin,et al.  The renaissance of fluorescence resonance energy transfer , 2000, Nature Structural Biology.

[3]  S. Weiss,et al.  Single-molecule fluorescence studies of protein folding and conformational dynamics. , 2006, Chemical reviews.

[4]  Serena Bernacchi,et al.  Excitonic heterodimer formation in an HIV-1 oligonucleotide labeled with a donor-acceptor pair used for fluorescence resonance energy transfer. , 2003, Biophysical journal.

[5]  Jeremy C. Smith,et al.  Fluorescence quenching of dyes by tryptophan: interactions at atomic detail from combination of experiment and computer simulation. , 2003, Journal of the American Chemical Society.

[6]  Sören Doose,et al.  Dynamics of unfolded polypeptide chains in crowded environment studied by fluorescence correlation spectroscopy. , 2007, Journal of molecular biology.

[7]  Ludwig Brand,et al.  Structural Characteristics of Fluorophores That Form Intramolecular H-Type Dimers in a Protease Substrate† , 1997 .

[8]  Shimon Weiss,et al.  Periodic acceptor excitation spectroscopy of single molecules , 2007, European Biophysics Journal.

[9]  T. Ha,et al.  Single-molecule fluorescence resonance energy transfer. , 2001, Methods.

[10]  M. Sauer,et al.  The initial step of DNA hairpin folding: a kinetic analysis using fluorescence correlation spectroscopy , 2006, Nucleic acids research.

[11]  Shammai Speiser,et al.  Photophysics and Mechanisms of Intramolecular Electronic Energy Transfer in Bichromophoric Molecular Systems: Solution and Supersonic Jet Studies. , 1996, Chemical reviews.

[12]  A. Deniz,et al.  Fluorescence quenching by TEMPO: a sub-30 A single-molecule ruler. , 2005, Biophysical journal.

[13]  M. Sauer,et al.  Using photoinduced charge transfer reactions to study conformational dynamics of biopolymers at the single-molecule level. , 2004, Current pharmaceutical biotechnology.

[14]  E. Elson,et al.  The kinetics of conformational fluctuations in an unfolded protein measured by fluorescence methods. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[15]  T Centner,et al.  Fluorescence quenching: A tool for single-molecule protein-folding study. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[16]  B. Packard,et al.  Profluorescent protease substrates: intramolecular dimers described by the exciton model. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[17]  Nam Ki Lee,et al.  Alternating‐Laser Excitation of Single Molecules , 2005 .

[18]  J. Steinfeld,et al.  Aggregation of equilibriums of xanthene dyes , 1972 .

[19]  M. Kasha,et al.  The exciton model in molecular spectroscopy , 1965 .

[20]  D. L. Dexter.  A Theory of Sensitized Luminescence in Solids , 1953 .

[21]  Nam Ki Lee,et al.  Accurate FRET measurements within single diffusing biomolecules using alternating-laser excitation. , 2005, Biophysical journal.

[22]  Nam Ki Lee,et al.  Fluorescence-aided molecule sorting: Analysis of structure and interactions by alternating-laser excitation of single molecules , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[23]  R. M. Cook,et al.  Intramolecular dimers: a new design strategy for fluorescence-quenched probes. , 2003, Chemistry.

[24]  R. Clegg.  Fluorescence resonance energy transfer and nucleic acids. , 1992, Methods in enzymology.

[25]  D. Neckers,et al.  Aggregation phenomena in xanthene dyes , 1989 .

[26]  M. Sauer,et al.  A close look at fluorescence quenching of organic dyes by tryptophan. , 2005, Chemphyschem : a European journal of chemical physics and physical chemistry.

[27]  X. Xie,et al.  Protein Conformational Dynamics Probed by Single-Molecule Electron Transfer , 2003, Science.

[28]  Ludwig Brand,et al.  Intramolecular Resonance Dipole−Dipole Interactions in a Profluorescent Protease Substrate , 1998 .

[29]  Sören Doose,et al.  A microscopic view of miniprotein folding: enhanced folding efficiency through formation of an intermediate. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[30]  T. Burghardt,et al.  Stereospecific reaction of muscle fiber proteins with the 5' or 6' isomer of (iodoacetamido)tetramethylrhodamine. , 1992, Biochemistry.

[31]  Shimon Weiss,et al.  The power and prospects of fluorescence microscopies and spectroscopies. , 2003, Annual review of biophysics and biomolecular structure.

[32]  Th. Förster.  Zwischenmolekulare Energiewanderung und Fluoreszenz , 1948 .

[33]  Christoph Bräuchle,et al.  Pulsed interleaved excitation. , 2005, Biophysical journal.