Commonly-used FRET fluorophores promote collapse of an otherwise disordered protein

The dimensions that unfolded and intrinsically disordered proteins (IDPs) adopt at low or no denaturant remains controversial. We recently developed an innovative analysis procedure for small-angle X-ray scattering (SAXS) profiles and found that even relatively hydrophobic IDPs remain nearly as expanded as the chemically denatured ensemble, rendering them significantly more expanded than is inferred from many fluorescence resonance energy transfer (FRET) studies. Here we show that fluor-phores typical of those added to IDPs for FRET studies contribute to this discrepancy. Specifically, we find that labeling a highly expanded IDP with Alexa488 causes its ensemble to contract significantly. We also tested the recent suggestion that FRET and SAXS results can be reconciled if, for unfolded proteins (and as opposed to the case for ideal random flight homopolymers), the radius of gyration (Rg) can vary independently from the chain’s end-to-end distance (Ree). Our analysis indicates, however, that SAXS is able to accurately extract Rg, ν and Ree even for heteropolymeric, protein-like sequences. From these studies we conclude that mild chain contraction and fluorophore-based interactions at lower denaturant concentrations, along with improved analysis procedures for both SAXS and FRET, can explain the preponderance of existing data regarding the nature of polypeptide chains unfolded in the absence of denaturant. Significance Statement Proteins can adopt a disordered ensemble, either prior to folding or as a part of their function. Simulations and fluorescence resonance energy transfer (FRET) studies often describe these disordered conformations as more compact than the fully random-coil state, whereas small-angle X-ray scattering studies (SAXS) indicate an expanded ensemble closely approximating the dimensions expected for the random coil. Resolving this discrepancy will enable more accurate predictions of protein folding and function. Here we reconcile these views by showing that the addition of common FRET fluorophores reduces the apparent dimensions of a disordered protein. Detailed analysis of both techniques, along with accounting for a moderate amount of fluorophore-induced contraction, demonstrates that disordered and unfolded proteins often remain well solvated and largely expanded in the absence of denaturant, properties that presumably minimize misfolding and aggregation.