TITLE: The photophysics of Förster resonance energy transfer studied by single-molecule spectroscopy

SPEAKER: Professor Chandran R. Sabanayagam

TIME: Thursday Sept. 22, 2005 at 4:00 PM

PLACE: George P. Williams, Jr. Lecture Hall, (Olin 101)

Förster resonance energy transfer (FRET) is the nonradiative dipole-dipole interaction between a donor and acceptor dye pair. The FRET efficiency is defined as E = (R_DA/R₀)⁶, where RDA is the interdye distance, and the constant R₀ is the Förster radius at which E is 50% . Unlike FRET experiments in bulk, with the single molecule approach the evolution of individual molecules can be followed over a broad range of times, from microseconds to minutes, making it especially useful for the study of biomolecular processes that involve complicated non-equilibrium kinetics. We developed an automated scanning confocal microscope to investigate the photophysics of single FRET pairs immobilized on a surface. By using B-DNA as a substrate, the donor and acceptor fluorophores were positioned at well-defined RDA values. The FRET efficiency was determined for various RDA ranging from 16Å to 58Å, and thousands of single-molecule spectra were collected for statistical analysis. Our experimentally determined values for E are in excellent agreement with theoretical predictions, and fits to E versus RDA yield R₀ constants that are similar to previously reported values. We demonstrate that single-molecule FRET can be used to quantify interdye distances, and that this technique can be used to determine real-time structural fluctuations of biomolecules.

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