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Effects of Fluorophore Attachment on Protein Conformation and Dynamics Studied by spFRET and NMR Spectroscopy
Author(s) -
SánchezRico Carolina,
Voith von Voithenberg Lena,
Warner Lisa,
Lamb Don C.,
Sattler Michael
Publication year - 2017
Publication title -
chemistry – a european journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.687
H-Index - 242
eISSN - 1521-3765
pISSN - 0947-6539
DOI - 10.1002/chem.201702423
Subject(s) - fluorophore , förster resonance energy transfer , chemistry , macromolecule , nuclear magnetic resonance spectroscopy , fluorescence , heteronuclear molecule , fluorescence spectroscopy , spectroscopy , intermolecular force , molecule , molecular dynamics , fluorescence in the life sciences , conjugated system , acceptor , computational chemistry , stereochemistry , polymer , organic chemistry , biochemistry , physics , quantum mechanics , condensed matter physics
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.