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Generation of an intramolecular three‐color fluorescence resonance energy transfer probe by site‐specific protein labeling
Author(s) -
Voss Stephanie,
Zhao Lei,
Chen Xi,
Gerhard Frank,
Wu YaoWen
Publication year - 2014
Publication title -
journal of peptide science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 66
eISSN - 1099-1387
pISSN - 1075-2617
DOI - 10.1002/psc.2590
Subject(s) - förster resonance energy transfer , chemistry , fluorescence , rhodamine , fluorescence in the life sciences , acceptor , fluorophore , intramolecular force , biophysics , stereochemistry , biology , physics , quantum mechanics , condensed matter physics
Fluorescence resonance energy transfer (FRET) is a valuable tool for studying protein structure, folding and interactions. The steep distance dependence of the FRET efficiency requires the donor and acceptor to be in close proximity (1–7.5 nm) to exhibit sufficient energy transfer. One possibility to overcome this limitation is the usage of a FRET cascade that utilizes more than one FRET pair. Essential for realizing this FRET cascade is the site‐specific introduction of multiple fluorophores to a given protein, which remains a great challenge. In this study, orthogonal labeling techniques, including fluorescent protein tagging, oxime ligation and kinetically controlled cysteine conjugation, are employed to introduce three fluorophores at specific sites of Rab1b GTPase, yielding a triple‐labeled FRET probe. The generated protein probe exhibits efficient energy transfer from the primary donor enhanced green fluorescent protein over the intermediate acceptor rhodamine to the final acceptor Dy630. The labeling strategy opens up a new avenue for multi‐color labeling of proteins, facilitating long‐distance FRET studies. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.