Saxs Confirms that FRET Dyes Promote Collapse of an Otherwise Fully Disordered Protein

Abstract

Significance: The dimensions of intrinsically disordered proteins (IDPs) remains controversial. Using SAXS, we previously found that even relatively hydrophobic IDPs remain expanded in the complete absence of denaturant, contrary to conclusions drawn from FRET studies that foldable sequences collapse in water. Here we use our methodology to demonstrate that labeling with Alexa488 causes the IDP to undergo significant dye-induced contraction. These results demonstrate that the addition of dyes influences the properties of IDPs and is likely the primary origin of the discrepancy between prior SAXS and FRET results. ABSTRACT: The origins of the discrepancy between SAXS and FRET measurements of IDP dimensions remain controversial. To address this, we recently developed a robust analysis for extracting Rg and ν (the Flory exponent; Rg∼Nν) from single SAXS experiments using a molecular form factor derived from simulations. Performing this analysis on a 334 AA IDP, we find that the addition of a dye pair promotes collapse, decreasing Rg from 51 to 45 Å and ν from 0.54 to 0.50. This observation is consistent with prior studies on PEG, which found the addition of dyes induces similar contraction1 despite the fact that, as we show here, the polymer behaves as a self-avoiding random walk. Recent studies have suggested that FRET and SAXS results can be reconciled if SAXS reports on Rg while remaining relatively insensitive to Rend-to-end, essentially decoupling the two. To the contrary, our analysis finds that the entire SAXS profile is sensitive to conformational ensembles enriched in shortened end-to-end distances, such as those produced by dye-dye interactions. More broadly, our simulations define a route to characterize the deviations from homopolymeric behavior inherent in disordered proteins. 1Watkins et al, PNAS 2015.