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Proceedings Paper

Heterogeneity and restricted state selection in FRET with fluorescent proteins
Author(s): T. S. Blacker; M. R. Duchen; A. J. Bain
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Paper 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.

Paper Details

Date Published: 1 March 2016
PDF: 16 pages
Proc. SPIE 9714, Single Molecule Spectroscopy and Superresolution Imaging IX, 971401 (1 March 2016); doi: 10.1117/12.2208895
Show Author Affiliations
T. S. Blacker, Univ. College London (United Kingdom)
M. R. Duchen, Univ. College London (United Kingdom)
A. J. Bain, Univ. College London (United Kingdom)

Published in SPIE Proceedings Vol. 9714:
Single Molecule Spectroscopy and Superresolution Imaging IX
Jörg Enderlein; Ingo Gregor; Zygmunt Karol Gryczynski; Rainer Erdmann; Felix Koberling, Editor(s)

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