Random coil negative control reproduces the discrepancy between scattering and FRET measurements of denatured protein dimensions

Abstract

Small-angle scattering studies generally indicate that the dimensions of unfolded single-domain proteins are independent (to within experimental uncertainty of a few percent) of denaturant concentration. In contrast, single-molecule FRET (smFRET) studies invariably suggest that protein unfolded states contract significantly as the denaturant concentration falls from high (∼6 M) to low (∼1 M). Here, we explore this discrepancy by using PEG to perform a hitherto absent negative control. This uncharged, highly hydrophilic polymer has been shown by multiple independent techniques to behave as a random coil in water, suggesting that it is unlikely to expand further on the addition of denaturant. Consistent with this observation, small-angle neutron scattering indicates that the dimensions of PEG are not significantly altered by the presence of either guanidine hydrochloride or urea. smFRET measurements on a PEG construct modified with the most commonly used FRET dye pair, however, produce denaturant-dependent changes in transfer efficiency similar to those seen for a number of unfolded proteins. Given the vastly different chemistries of PEG and unfolded proteins and the significant evidence that dye-free PEG is well-described as a denaturant-independent random coil, this similarity raises questions regarding the interpretation of smFRET data in terms of the hydrogen bond-or hydrophobically driven contraction of the unfolded state at low denaturant.protein folding | SAXS | two state | statistical coil | Flory scaling R ecent years have seen a significant controversy arise regarding the behavior of the unfolded states of single-domain proteins in response to changing levels of chemical denaturant. That is, although small-angle X-ray scattering (SAXS) and single-molecule FRET (smFRET) results are all consistent with the argument that, at high levels of chemical denaturant (∼6 M or above), unfolded proteins adopt a swollen, self-avoiding ensemble well-approximated as an excluded volume random coil (1-3), the two approaches produce seemingly discrepant results for the dimensions of the unfolded states populated at lower (∼1 M) denaturant (4). For example, time-resolved SAXS studies of the unfolded state transiently populated when protein L is rapidly shifted from high guanidine hydrochloride (GuHCl) to low denaturant conditions produce no experimentally significant evidence for the compaction of this single-domain protein before folding (4, 5) [e.g., estimated radii of gyration of 25.1 ± 0.3 Å for the unfolded state at equilibrium in 7 M GuHCl and 24.9 ± 1.1 Å for the transient unfolded state populated at 0.67 M GuHCl; confidence intervals are SEs (4)]. In contrast, multiple smFRET studies conducted at equilibrium suggest that the unfolded state of dye-labeled protein L contracts by 20-40% over this same range of denaturant concentrations (6, 7) (Fig.

Decoupling of size and shape fluctuations in heteropolymeric sequences reconciles discrepancies in SAXS vs. FRET measurements

“…This is evidenced by larger values of α(R E,L ) vs. smaller values of α(R G,L ) (on average 2.02 ± 0.18 vs. 1.27 ± 0.12, respectively; individual values given in SI Appendix, Table S9). These findings are concordant with previous results, which point to disagreements between inferences from SAXS/small-angle neutron scattering (SAXS/SANS) and smFRET measurements at low denaturant concentrations (15,21). SAXS measurements of labeled vs. unlabeled molecules rule out the dyes as the source of the discrepancy.…”

“…Therefore, one might conclude that the collapse transition is virtually nonexistent for IDPs and abrupt and concomitant with the ratelimiting folding transition for autonomously folding proteins. The discrepancies in interpretations regarding the collapse transition for protein folding and for IDPs have led to numerous debates (4,9,15,(20)(21)(22)(23).…”

“…Both techniques have distinct strengths and weaknesses (15,(20)(21)(22)(23). Strengths of smFRET measurements include the ultralow protein concentrations, at which experiments can be conducted, the ability to resolve distinct conformational populations, and the advantage of following motions across timescales that range from the nanosecond to the millisecond regimes.…”

“…The discrepant inferences drawn from SAXS and smFRET measurements have stimulated numerous debates and independent investigations. Discrepancies were recently reported for nonbiological homopolymers like polyethylene glycol (PEG) (21). This study compared R G values from SANS experiments to R E values derived from smFRET.…”

Förster resonance energy transfer: Role of diffusion of fluorophore orientation and separation in observed shifts of FRET efficiency

“…We consider how such kinetic mechanisms relate to some recent experiments investigating the origins of shifts in FRET efficiency [19, 34, 35, 58]. …”