Fully Reduced Ribonuclease A Does not Expand at High Denaturant Concentration or Temperature

Jacob, J.; Dothager, Robin S.; Thiyagarajan, P.; Sosnick, Tobin R.

doi:10.1016/j.jmb.2007.01.012

Cited by 40 publications

(47 citation statements)

References 76 publications

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“…Although it is widely accepted that chemically denatured proteins adopt an ensemble of conformations well-described as random coil (2), significant debate remains regarding the nature of the unfolded states populated at low denaturant concentrations (4). Specifically, time-resolved SAXS measurements collected within milliseconds of transfer from high to low denaturant suggest that chemically denatured single-domain proteins do not undergo any significant conformational change before folding itself (5,(12)(13)(14). (Left) Shown, for example, are SAXS profiles collected on protein L at equilibrium at 1 M (native conditions), at equilibrium at 4 M GuHCl (unfolding conditions), and transiently before folding on transfer to denaturant concentrations as low as 0.67 M; the near-superimposability of the scaled scattering data for the various unfolded states suggests that the unfolded chain does not change dimensions (to within experimental uncertainty of a few percent) before folding (4).…”

Section: Resultsmentioning

confidence: 99%

“…Because the pertinent SAXS experiments fail to produce evidence of unfolded-state contraction, the most relevant controls for these studies are positive controls establishing the technique's ability to detect unfolded-state compaction, if it were to occur, of the magnitude seen by smFRET. Fortunately, these controls are already in hand: SAXS has repeatedly been shown to distinguish (at confidence intervals of many σ) between the dimensions of chemically unfolded, disulfide-free proteins, which generally coincide closely with expected random coil dimensions (2), and the 20-30% more compact (but still flexible and unfolded) states seen when the same chemically denatured protein is cross-linked through disulfide bonds (11,12,34,35). Conversely, because smFRET studies of unfolded states are universally interpreted in terms of significant collapse, the most critical controls for smFRET would be negative controls (that is, the demonstration of the denaturant independence of smFRET across an unfolded polymer, the dimensions of which are known to be denaturant-independent).…”

Section: Significancementioning

confidence: 94%

“…1). Moreover, this discrepancy seems to be nearly universal among single-domain proteins: whereas the results of at least a half-dozen SAXS studies on chemically unmodified, single-domain proteins fail to detect any experimentally significant evidence (at experimental precision of, typically, a few percent) of contraction (8)(9)(10)(11)(12)(13)(14)(15), all of the more than one-dozen smFRET studies of dye-labeled, singledomain proteins reported to date have been interpreted in terms of unfolded states that contract as the denaturant concentration is lowered to ∼1 M (2,6,7,(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26).…”

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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: Resultsmentioning

confidence: 99%

Section: Significancementioning

confidence: 94%

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confidence: 99%

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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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“…This equivalence indicates that the two methods probe the same folding transition to a DSE that is devoid of stable H bonds. Moreover, data from small-angle X-ray scattering imply that the dimensions in the DSE of many [but not all (56,57)] small proteins remain unchanged upon shifting to a lower level of denaturant (32,(58)(59)(60). Furthermore, most [but not all (61)] small proteins satisfy the two-state chevron criterion, sometimes even down to 0 M denaturant (21,22), indicating that the DSE remains highly solvated under native conditions for many proteins.…”

Section: Discussionmentioning

confidence: 99%

Cooperative folding near the downhill limit determined with amino acid resolution by hydrogen exchange

Baxa

Gagnon

et al. 2016

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

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The relationship between folding cooperativity and downhill, or barrier-free, folding of proteins under highly stabilizing conditions remains an unresolved topic, especially for proteins such as λ-repressor that fold on the microsecond timescale. Under aqueous conditions where downhill folding is most likely to occur, we measure the stability of multiple H bonds, using hydrogen exchange (HX) in a λ YA variant that is suggested to be an incipient downhill folder having an extrapolated folding rate constant of 2 × 10 5 s −1 and a stability of 7.4 kcal·mol −1 at 298 K. At least one H bond on each of the three largest helices (α1, α3, and α4) breaks during a common unfolding event that reflects global denaturation. The use of HX enables us to both examine folding under highly stabilizing, native-like conditions and probe the pretransition state region for stable species without the need to initiate the folding reaction. The equivalence of the stability determined at zero and high denaturant indicates that any residual denatured state structure minimally affects the stability even under native conditions. Using our ψ analysis method along with mutational ϕ analysis, we find that the three aforementioned helices are all present in the folding transition state. Hence, the free energy surface has a sufficiently high barrier separating the denatured and native states that folding appears cooperative even under extremely stable and fast folding conditions. T he nature of the energy landscape when folding occurs on the microsecond timescale continues to be investigated using both experimental and computational approaches. The 80-residue subdomain of the λ-repressor transcription factor, λ 6-85 (1-11), is an appealing system as it contains five α-helices arranged in a nonsymmetric pattern, folds in microseconds, and is nearly a downhill folder (12), a condition where a continuous series of folding events may be characterized. These characteristics along with a folding time that nears the upper limit for current all-atom simulations (13-19) make this protein appropriate for detailed comparisons between experiment and simulations.The energy landscape of λ 6-85 is significantly influenced by its sequence. Oas and coworkers found that the wild type and a fasterfolding mutant with two helix-promoting substitutions (λ AA with G46A/G48A, Fig. S1A) fold cooperatively (1-4). The transition state ensemble (TSE) characterized using ϕ analysis minimally contains α1 and α4 (3). Kinetic H/D isotope effect measurements in the Sosnick laboratory found that both λ 6-85 and λ AA fold cooperatively with ∼70% of the H bonds being formed in the TSE, matching the degree of surface buried in the TSE [beta Tanford value (β Tanford )] (5).Using nanosecond T-jump and other methods, Gruebele and coworkers proposed that faster-folding and stabilized variants, such as λ YA (λ AA + Q33Y) and λ D14A (λ YA + D14A), fold in an incipient or fully downhill manner, especially under highly stable conditions at lower temperatures (6-9). Key signatures of downhi...

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“…[11][12][13][14][15][16][17][18] In addition, several single-molecule fluorescence resonance energy transfer studies 26,29,32 have shown that conformational transitions within an unfolded protein ensemble could occur on a range of timescales, signifying the underlying hierarchical nature of the unfolded potential well. While these previous studies provided valuable insights into the statistical properties of unfolded polypeptide chains, they mainly focused on relatively small and mostly single-domain proteins.…”

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Denaturant-induced Expansion and Compaction of a Multi-domain Protein: IgG

Guo

Chowdhury

Glasscock

et al. 2008

Journal of Molecular Biology

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It is generally believed that unfolded or denatured proteins show random coil statistics and hence their radius of gyration simply scales with solvent quality (or denaturant concentration). Indeed, nearly all proteins studied thus far have been shown to undergo a gradual and continuous expansion with increasing denaturant concentration. Here, we use fluorescence correlation spectroscopy (FCS) to show that while protein A, a multidomain and predominantly helical protein, expands gradually and continuously with increasing concentration of guanidine hydrochloride (GdnHCl), the F(ab′) 2 fragment of goat anti-rabbit antibody IgG, a multi-subunit all β-sheet protein, however, does not show such continuous expansion behavior. Instead, it first expands and then contracts with increasing GdnHCl concentration. Even more striking is the fact that the hydrodynamic radius of the most expanded F(ab′) 2 ensemble, observed at 3-4 M GdnHCl, is ca. 3.6 times that of the native protein.Further FCS measurements involving urea and NaCl show that the unusually expanded F(ab′) 2 conformations might be due to electrostatic repulsions. Taken together, these results suggest that specific interactions need to be considered while assessing the conformational and statistical properties of unfolded proteins, particularly under conditions of low solvent quality. KeywordsFCS; Flory's random coil model; IgG; protein folding; unfolded state Despite its obvious importance in understanding how proteins fold, the unfolded or denatured state of proteins remains relatively unexplored. 1,2 Recent years have thus seen an increasing number of studies focused on the conformational properties of proteins in their unfolded state. 3-24 Of particular interest are those which assess the molecular dimensions as well as conformational dynamics of proteins under various denaturing conditions using ensemble 11-20 or single-molecule techniques. 25-32 All these studies, while involving a wide variety of peptides and proteins that span a large range of chain lengths (8-549 aa), generally support the notion that unfolded proteins behave as self-avoiding random-coils which undergo a continuous expansion with increasing denaturant concentration, following Flory's powerlaw relationship (i.e., R G = R 0 N ν ; where R G is the radius of gyration, R 0 is a constant determined *Corresponding author. Email address: gai@sas.upenn.edu. & L.G. and P.C. contributed equally to this work.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Figure 1a). Because of its distinctive structural characteristics and the prevalence of antibodies...

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Fully Reduced Ribonuclease A Does not Expand at High Denaturant Concentration or Temperature

Cited by 40 publications

References 76 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

Cooperative folding near the downhill limit determined with amino acid resolution by hydrogen exchange

Denaturant-induced Expansion and Compaction of a Multi-domain Protein: IgG

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