Measuring distances within unfolded biopolymers using fluorescence resonance energy transfer: The effect of polymer chain dynamics on the observed fluorescence resonance energy transfer efficiency

Makarov, Dmitrii E.; Plaxco, Kevin W.

doi:10.1063/1.3212602

Cited by 25 publications

(30 citation statements)

References 41 publications

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“…This similarity raises questions regarding the interpretation of such changes as being indicative of hydrophobic or hydrogen bond-driven collapse. likewise, been shown to be too small to account for the seeming inconsistency between SAXS-and FRET-based observations (33). Again, the source of this experimentally robust discrepancy remains stubbornly unclear (a more detailed discussion of this issue is in ref.…”

Section: Significancementioning

confidence: 96%

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

Watkins

Simon

Sosnick

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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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.

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Section: Significancementioning

confidence: 96%

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

Watkins

Simon

Sosnick

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

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“…S6). Case C. It is known that the dynamics of dyes can affect E FRET measurements (9,31,33,(59)(60)(61)(62)(63). For unfolded proteins of similar size and in similar solvents to the ones studied here (including NUS), chain reconfiguration times have been shown to be in the range of ∼100 ns (3,64), which is well above the donor lifetimes of ∼4 ns and well below the transit times through the confocal volume, ∼1 ms. As a result, a major role of dynamics in the measured intensity-based E FRET values seems unlikely.…”

mentioning

confidence: 88%

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

Fuertes

Banterle

Ruff

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

210

313

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Unfolded states of proteins and native states of intrinsically disordered proteins (IDPs) populate heterogeneous conformational ensembles in solution. The average sizes of these heterogeneous systems, quantified by the radius of gyration (R G ), can be measured by small-angle X-ray scattering (SAXS). Another parameter, the mean dye-to-dye distance (R E ) for proteins with fluorescently labeled termini, can be estimated using single-molecule Förster resonance energy transfer (smFRET). A number of studies have reported inconsistencies in inferences drawn from the two sets of measurements for the dimensions of unfolded proteins and IDPs in the absence of chemical denaturants. These differences are typically attributed to the influence of fluorescent labels used in smFRET and to the impact of high concentrations and averaging features of SAXS. By measuring the dimensions of a collection of labeled and unlabeled polypeptides using smFRET and SAXS, we directly assessed the contributions of dyes to the experimental values R G and R E . For chemically denatured proteins we obtain mutual consistency in our inferences based on R G and R E , whereas for IDPs under native conditions, we find substantial deviations. Using computations, we show that discrepant inferences are neither due to methodological shortcomings of specific measurements nor due to artifacts of dyes. Instead, our analysis suggests that chemical heterogeneity in heteropolymeric systems leads to a decoupling between R E and R G that is amplified in the absence of denaturants. Therefore, joint assessments of R G and R E combined with measurements of polymer shapes should provide a consistent and complete picture of the underlying ensembles.single-molecule FRET | intrinsically disordered proteins | denatured-state ensemble | protein folding | polymer theory Q uantitative characterizations of the sizes, shapes, and amplitudes of conformational fluctuations of unfolded proteins under denaturing and native conditions are directly relevant to advancing our understanding of the collapse transition during protein folding. These types of studies are also relevant to furthering our understanding of the functions and interactions of intrinsically disordered proteins (IDPs) in physiologically relevant conditions (1). Polymer physics theories provide the conceptual foundations for analyzing conformationally heterogeneous systems such as IDPs and unfolded ensembles of autonomously foldable proteins (2-4). Specifically, order parameters in theories of coil-toglobule transitions and analytical descriptions of conformational ensembles (5, 6) are based on ensemble-averaged values of radii of gyration (R G ) and amplitudes of fluctuations measured by end-toend distances (R E ).Estimates of R G are accessible through small-angle X-ray scattering (SAXS) measurements because scattering intensities are directly related to the global protein size (Fig. 1) (7, 8). At finite concentrations, assuming the absence of intermolecular interactions, R G is proportional to the square root o...

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“…These measurements were not used for calculating mean distance, since the ensemble heterogeneity and chain dynamics contribute to the measured energy transfer efficiencies 40,53 ; thus, distance calculations were obtained only from global analysis of the trFRET measurements, which can dissect the effects of conformational heterogeneity and chain dynamics. Both the steady-state and the time-resolved FRET measurements were done using a protein concentration at or below the critical concentration of ∼ 20 μM.…”

Section: Steady-state Fret Measurementsmentioning

confidence: 99%

“…39,40 In the present study, we simultaneously determined the parameters describing the distributions of intramolecular distances and intramolecular diffusion coefficients of segment ends by application of trFRET measurements of double-labeled αS mutants.…”

Section: Introductionmentioning

confidence: 99%

Segmental Conformational Disorder and Dynamics in the Intrinsically Disordered Protein α-Synuclein and Its Chain Length Dependence

Grupi

Haas

2011

Journal of Molecular Biology

112

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Measuring distances within unfolded biopolymers using fluorescence resonance energy transfer: The effect of polymer chain dynamics on the observed fluorescence resonance energy transfer efficiency

Cited by 25 publications

References 41 publications

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

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

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

Segmental Conformational Disorder and Dynamics in the Intrinsically Disordered Protein α-Synuclein and Its Chain Length Dependence

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