Commonly used FRET fluorophores promote collapse of an otherwise disordered protein

Abstract

The dimensions that unfolded proteins, including intrinsically disordered proteins (IDPs), adopt in the absence of denaturant remain controversial. We developed an analysis procedure for small-angle X-ray scattering (SAXS) profiles and used it to demonstrate that even relatively hydrophobic IDPs remain nearly as expanded in water as they are in high denaturant concentrations. In contrast, as demonstrated here, most fluorescence resonance energy transfer (FRET) measurements have indicated that relatively hydrophobic IDPs contract significantly in the absence of denaturant. We use two independent approaches to further explore this controversy. First, using SAXS we show that fluorophores employed in FRET can contribute to the observed discrepancy. Specifically, we find that addition of Alexa-488 to a normally expanded IDP causes contraction by an additional 15%, a value in reasonable accord with the contraction reported in FRET-based studies. Second, using our simulations and analysis procedure to accurately extract both the radius of gyration (Rg) and end-to-end distance (Ree) from SAXS profiles, we tested the recent suggestion that FRET and SAXS results can be reconciled if the Rg and Ree are “uncoupled” (i.e., no longer simply proportional), in contrast to the case for random walk homopolymers. We find, however, that even for unfolded proteins, these two measures of unfolded state dimensions remain proportional. Together, these results suggest that improved analysis procedures and a correction for significant, fluorophore-driven interactions are sufficient to reconcile prior SAXS and FRET studies, thus providing a unified picture of the nature of unfolded polypeptide chains in the absence of denaturant.

Integrating multiple experimental data to determine conformational ensembles of an intrinsically disordered protein

“…Analysing the SAXS data with the homopolymer MFF approach [34,38] resulted in ν app which is very similar to the ν app obtained by globally fitting the ISPs with fixed l p (SI Appendix, Table S6 ). Using a heterpolymer MFF analysis, which allows for deviations in power-law scaling at long sequence separations (MFF-het3, SI Appendix, Table S6 ), gave similar results to separately fitting ISPs at intermediate and longsequence separations (ν app = 0.56 ± 0.01 and ∆ν ends app = −0.17 ± 0.08 for Sic1).…”

“…While attractive fluorophore interactions have been used to explain the smFRET technique's discrepant inferences for IDP ensembles relative to SAXS [38], we consider whether optimization using multiple solution data types might reduce or eliminate such discrepancies. One solution to reduce or eliminate the apparent smFRET and SAXS discrepancy is to jointly restrain ensembles with both data sets simultaneously [27,35].…”

“…Similarly, ISPs explain how R ee and R g can become decoupled for finite-length heteropolymers [27,38].…”

“…3 A is consistent with the entirety of the experimental data on Sic1. We therefore consider this ISP as the benchmark in determining the scaling behaviour of the Sic1 ensemble and compare it to recently published methods which infer scaling behaviour from only SAXS, or only smFRET data [34,38,50].…”

“…Similarly, Riback and coworkers have recently introduced a procedure for fitting SAXS data by pregenerating ensembles of conformations with different properties (specifically, the strength and patterning of inter-residue attractions) and extracting dimensionless "molecular form factors" (MFFs) [34,38]. The properties of interest are then inferred from the ensemble whose MFF best fits the data.…”