Evaluation of peptide fluorescence quenching by thiourea. A) Relative fluorescence units (RFUs, λex =494, λem =520 nm) of 10 μL of 10 nM FITC- or FAM-conjugated 4E-BP1 peptides in assay buffer (50 mM sodium phosphate pH 7.4, 200 mM NaCl, 1 mM DTT, and 1 mM EDTA) in the absence or in the presence of 50 % TFE. FAM-conjugated peptide fluorescence does not increase in TFE to the same degree as that of the FITC-conjugated peptide. Fluorescence measurements were made in triplicate; p values were generated in GraphPad prism by using two-way ANOVA. *** p =0.001; ** p =0.004. B) FITC-and FAM-conjugated 4E-BP1 peptides exhibit similar increases in helicity in the presence of TFE, as determined by CD spectroscopy (100 μM peptide in 50 mM sodium phosphate buffer, pH 7.4) with or without 50 % TFE at 25 °C.