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

Gauging the flexibility of fluorescent markers for the interpretation of fluorescence resonance energy transfer
Gauging the flexibility of fluorescent markers for the interpretation of fluorescence resonance energy transfer
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
0002-7863
279-285
Rindermann, Jan J.
c69ba4d0-0a39-4cf8-a163-6e1e731c9fd4
Akhtman, Yosef
955350e0-6c75-4617-b666-743f35221bd1
Richardson, James
51db9f73-a136-48af-8722-21d46906cf40
Brown, Tom
a64aae36-bb30-42df-88a2-11be394e8c89
Lagoudakis, Pavlos G.
ea50c228-f006-4edf-8459-60015d961bbf
Rindermann, Jan J.
c69ba4d0-0a39-4cf8-a163-6e1e731c9fd4
Akhtman, Yosef
955350e0-6c75-4617-b666-743f35221bd1
Richardson, James
51db9f73-a136-48af-8722-21d46906cf40
Brown, Tom
a64aae36-bb30-42df-88a2-11be394e8c89
Lagoudakis, Pavlos G.
ea50c228-f006-4edf-8459-60015d961bbf

Rindermann, Jan J., Akhtman, Yosef, Richardson, James, Brown, Tom and Lagoudakis, Pavlos G. (2011) Gauging the flexibility of fluorescent markers for the interpretation of fluorescence resonance energy transfer. Journal of the American Chemical Society, 133 (2), 279-285. (doi:10.1021/ja105720j).

Record type: Article

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

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Published date: 2011

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Local EPrints ID: 179809
URI: http://eprints.soton.ac.uk/id/eprint/179809
ISSN: 0002-7863
PURE UUID: 5fdce93e-6ceb-46de-9c99-487ec7905d0f

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Date deposited: 13 Apr 2011 09:11
Last modified: 25 Nov 2019 21:16

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