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

Fluorescence-based techniques are widely used to study biomolecular conformations, intra- and intermolecular interactions, and conformational dynamics of macromolecules. Especially for fluorescent-based single-molecule experiments, the choice of the fluorophore and labeling position are highly important. Here, we have 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. We show 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. We find that 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.