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Sanchez-Rico, C. ; VoithvonVoithenberg, L.* ; Warner, L. ; Lamb, D.C.* ; Sattler, M.

Effects of fluorophore attachment on protein conformation and dynamics studied by spFRET and NMR spectroscopy.

Chem. Eur. J. 23, 14267-14277 (2017)
Verlagsversion DOI
Open Access Green möglich sobald Postprint bei der ZB eingereicht worden ist.
Fluorescence-based techniques are widely used to study biomolecular conformations, intra- and intermolecular interactions, and conformational dynamics of macromolecules. Especially for fluorescence-based single-molecule experiments, the choice of the fluorophore and labeling position are highly important. In this work, we studied the biophysical and structural effects that are associated with the conjugation of fluorophores to cysteines in the splicing factor U2AF65 by using single pair Förster resonance energy transfer (FRET) and nuclear magnetic resonance (NMR) spectroscopy. It is shown that certain acceptor fluorophores are advantageous depending on the experiments performed. The effects of dye attachment on the protein conformation were characterized using heteronuclear NMR experiments. The presence of hydrophobic and aromatic moieties in the fluorophores can significantly affect the conformation of the conjugated protein, presumably by transient interactions with the protein surface. Guidelines are provided for carefully choosing fluorophores, considering their photophysical properties and chemical features for the design of FRET experiments, and for minimizing artifacts.
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Publikationstyp Artikel: Journalartikel
Dokumenttyp Wissenschaftlicher Artikel
Schlagwörter Fluorophores ; Fret ; Multidomain Proteins ; Nmr Spectroscopy ; Protein-rna Interactions; Single-molecule Fret; Dyes Intramolecular Stabilization; Spin Labels; Organic Fluorophores; Fluorescent Proteins; Energy-transfer; Cell; Probes; Dna; Microscopy
ISSN (print) / ISBN 0947-6539
e-ISSN 1521-3765
Quellenangaben Band: 23, Heft: 57, Seiten: 14267-14277 Artikelnummer: , Supplement: ,
Verlag Wiley
Verlagsort Weinheim
Begutachtungsstatus Peer reviewed