Inferring Properties of Disordered Chains From FRET Transfer Efficiencies

Abstract

Förster resonance energy transfer (FRET) is a powerful tool for elucidating both structural and dynamic properties of unfolded or disordered biomolecules, especially in single-molecule experiments. However, the key observables, namely, the mean transfer efficiency and fluorescence lifetimes of the donor and acceptor chromophores, are averaged over a broad distribution of donor-acceptor distances. The inferred average properties of the ensemble therefore depend on the form of the model distribution chosen to describe the distance, as has been widely recognized. In addition, while the distribution for one type of polymer model may be appropriate for a chain under a given set of physico-chemical conditions, it may not be suitable for the same chain in a different environment so that even an apparently consistent application of the same model over all conditions may distort the apparent changes in chain dimensions with variation of temperature or solution composition. Here, we present an alternative and straightforward approach to determining ensemble properties from FRET data, in which the polymer scaling exponent is allowed to vary with solution conditions. In its simplest form, it requires either the mean FRET efficiency or fluorescence lifetime information. In order to test the accuracy of the method, we have utilized both synthetic FRET data from implicit and explicit solvent simulations for 30 different protein sequences, and experimental single-molecule FRET data for an intrinsically disordered and a denatured protein. In all cases, we find that the inferred radii of gyration are within 10% of the true values, thus providing higher accuracy than simpler polymer models. In addition, the scaling exponents obtained by our procedure are in good agreement with those determined directly from the molecular ensemble. Our approach can in principle be generalized to treating other ensemble-averaged functions of intramolecular distances from experimental data. Förster resonance energy transfer (FRET) is a powerful tool for elucidating both structural and dynamic properties of unfolded or disordered biomolecules, especially in single-molecule experiments. However, the key observables, namely the mean transfer efficiency and fluorescence lifetimes of the donor and acceptor chromophores, are averaged over a broad distribution of donor-acceptor distances. The inferred average properties of the ensemble therefore depend on the form of the model distribution chosen to describe the distance, as has been widely recognized. In addition, while the distribution for one type of polymer model may be appropriate for a chain under a given set of physico-chemical conditions, it may not be suitable for the same chain in a different environment, so that even an apparently consistent application of the same model over all conditions may distort the apparent changes in chain dimensions with variation of temperature or solution composition. Here, we present an alternative and straightforward approach to determining ensemble proper...

Commonly used FRET fluorophores promote collapse of an otherwise disordered protein

“…In combination with prior studies (9), similar conclusions can be inferred for PEG, a known SARW. These findings, along with our prior result that disordered chains undergo a mild expansion in denaturant (45) and improved methods for extracting R g values from FRET data (40,(42)(43)(44), now provide a sufficient framework for resolving discrepancies between SAXS and FRET on the dimensions of disordered proteins. The fundamental and significant conclusion of the resulting unified picture is that, even in the absence of denaturant, water remains a good solvent for most unfolded proteins.…”

“…The application of more realistic simulations and analytical models resulted in FRET-derived distances having a smaller denaturant dependence (Fig. 1A, Bottom) (40,(42)(43)(44). In parallel, improved SAXS data and analysis, including the use of the dimensionless Kratky plot to emphasize changes in ν rather than R g , (which is important, as the addition of fluorophores near the ends of a chain will increase R g due to their mass), likewise provided evidence for a minor contraction below 2 M Gdn (Fig.…”

An Extended Guinier Analysis for Intrinsically Disordered Proteins

“…This would seem to make the fitting scheme less robust due to the already limited number of experimental data points in the relevant range of q for many IDPs. However, encouraged by our recent success in using a self-avoiding walk (SAW) model[44, 45] to interpret experimental FRET data from IDPs[43], we sought to introduce a dependence between R g and ν based on the SAW,…”

“…in which γ ≈ 1.1615 [52], the prefactor b has been determined to be 0.55 nm for proteins [14, 43] and N is the number of peptide bonds in the chain (i.e. one less than the number of residues).…”

“…This motivates us to introduce an empirical fourth order term dependent on ν . Similar to our recent data interpretation method for FRET [43], in which both the end-end distance and ν were determined simultaneously by introducing the correlation between the end-end distance distribution and ν using a self-avoiding walk model [44, 45, 46], we introduce the correlation between R g and ν using the same polymer model. Together with the empirically determined fourth order term, we have developed an extended version of Guinier analysis in which both R g and ν are fitted at the same time and no additional free parameters are introduced.…”