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Heterogeneity and restricted state selection in FRET with fluorescent proteins

Bain, AJ; Blacker, TS; Duchen, M; (2016) Heterogeneity and restricted state selection in FRET with fluorescent proteins. In: Single Molecule Spectroscopy and Superresolution Imaging IX. Society of Photo-Optical Instrumentation Engineers (SPIE): San Francisco, CA, USA. Green open access

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Abstract

Most fluorescent proteins exhibit multi-exponential fluorescence decays, indicating the presence of a heterogeneous excited state population. In the analysis of FRET to and between fluorescent proteins, it is often convenient to assume that a single interaction pathway is involved. However, in recent work we have shown that this assumption does not hold. Moreover, certain pathways can be highly constrained, leading to the potential misinterpretation of experimental data concerning protein-protein interactions. FRET and single-photon absorption both obey the same global electric dipole selection rules but differ greatly in the mechanism of the acceptor photoselection. In an isotropic medium, single-photon excitation accesses all acceptor transition dipole moment orientations with an equal probability. However, the FRET rate depends on the relative orientation of the donor and acceptor through the κ2 orientation parameter. We show how time- and spectrally- resolved fluorescence intensity and anisotropy decay measurements following direct acceptor excitation, combined with those of the interacting FRET pair, can be used to identify restricted FRET state selection and thus provide accurate measurements of protein-protein interaction dynamics.

Type: Proceedings paper
Title: Heterogeneity and restricted state selection in FRET with fluorescent proteins
Event: Single Molecule Spectroscopy and Superresolution Imaging IX
Location: San Francisco
Open access status: An open access version is available from UCL Discovery
DOI: 10.1117/12.2208895
Publisher version: http://dx.doi.org/10.1117/12.2208895
Language: English
Additional information: Copyright © 2016 Society of Photo Optical Instrumentation Engineers. One print or electronic copy may be made for personal use only. Systematic reproduction and distribution, duplication of any material in this paper for a fee or for commercial purposes, or modification of the content of the paper are prohibited.
Keywords: Fluorescence resonance energy transfer; Resonance energy transfer; Fluorescent proteins; Proteins; Single photon; Luminescence; Absorption; Fluorescence anisotropy
UCL classification: UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Cell and Developmental Biology
UCL > Provost and Vice Provost Offices > UCL BEAMS
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences > Dept of Physics and Astronomy
URI: http://discovery.ucl.ac.uk/id/eprint/1494313
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