Solvent effect on FRET spectroscopic ruler.

A discrepancy has emerged in recent years between single-molecule Förster resonance energy transfer (smFRET) measurements and small angle X-ray scattering (SAXS) or small angle neutron scattering experiments in the study of unfolded or intrinsically disordered proteins in denaturing solutions. Despite significant advances that have been made in identifying various factors which may have contributed to the manifestation of the so-called smFRET-SAXS discrepancy, no consensus has been reached so far on its original source or eventual resolution. In this study, we investigate this problem from the perspective of the solvent effect on FRET spectroscopic ruler (SEFSR), a generic term we use to describe various solvent-dependent factors affecting the accuracy of the FRET experimental method that is known as a "spectroscopic ruler." Some factors belonging to SEFSR, such as direct dye-solvent interaction and labeling configuration, seem to have not received due attention regarding their significance in contributing to the discrepancy. We identify SEFSR by measuring a rigid segment of a double-stranded DNA in various solutions using the smFRET method and evaluate its relative importance in smFRET experiments by measuring segments of a single-stranded DNA and polyethylene glycol (PEG) in solutions. We find that SEFSR can produce non-negligible FRET-inferred interdye distance changes in various solutions, with an intensity following the Hofmeister series in ionic solutions and dependent on labeling configurations. SEFSR is found to be significant in GuHCl and urea solutions, which can fully cover the apparent expansion signal of dye-labeled PEG. Our findings suggest that SEFSR may have played an important role in contributing to the smFRET-SAXS discrepancy.