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Chemistry. 2017 Oct 12;23(57):14267-14277. doi: 10.1002/chem.201702423. Epub 2017 Sep 12.

Effects of Fluorophore Attachment on Protein Conformation and Dynamics Studied by spFRET and NMR Spectroscopy.

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Institute of Structural Biology, Helmholtz Zentrum München, Ingolstädter Landstr. 1, 85764, Neuherberg, Germany.
Biomolecular NMR and Center for Integrated Protein Science Munich at Department of Chemistry, Technical University of Munich, Lichtenbergstr. 4, 85747, Garching, Germany.
Physical Chemistry, Department of Chemistry, Munich Center for Integrated Protein Science, Nanosystems Initiative Munich and Centre for Nanoscience, Ludwig-Maximilians-Universität München, Butenandtstr. 5-13, 81377, Munich, Germany.


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.


FRET; NMR spectroscopy; fluorophores; multidomain proteins; protein-RNA interactions

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