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Journal of the American Chemical Society 133 (2), 279–285 (2011)

posted 13 Feb 2011, 06:00 by Pavlos Lagoudakis   [ updated 13 Feb 2011, 06:23 ]

"Gauging the flexibility of fluorescent markers for the interpretation of fluorescence resonance energy transfer"

Journal of the American Chemical Society 133 (2), 279–285 (2011)

Abstract: Intramolecular distances in proteins and other biomolecules can be studied in the living cell by means of fluorescence resonance energy transfer (FRET) in steady state or pulsed-excitation experiments. The major uncertainty originates from the unknown orientation between the optical dipole moments of the fluorescent markers, especially when the molecule undergoes thermal fluctuations in physiological conditions. We introduce a statistical method for the interpretation of fluorescence decay dynamics in donor-acceptor FRET pairs that allows us to retrieve both the orientation and the extent of directional fluctuations of the involved dipole moments. We verify the method by applying it to donor-acceptor pairs controllably attached to DNA helices and find that common assumptions such as complete rotational freedom or fully hindered rotation of the dipoles fail a physical interpretation of the fluorescence decay dynamics. This methodology is applicable in single molecule and ensemble measurements of FRET to derive more accurate distance estimates from optical experiments, without the need for more complex and expensive NMR studies.