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

Jan J. Rindermann, Yosef Akhtman, James Richardson, Tom Brown, Pavlos G. Lagoudakis

Research output: Contribution to journalArticlepeer-review

18 Citations (Scopus)

Abstract

Intramolecular distances in proteins and other biomolecules can be studied in living cells 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 based on the von Mises-Fisher distribution 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.

Original languageEnglish
Pages (from-to)279-285
Number of pages7
JournalJournal of the American Chemical Society
Volume133
Issue number2
DOIs
Publication statusPublished - 19 Jan 2011
Externally publishedYes

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