Structure of proteins unfolded by guanidinium chloride

Calmettes, P.; Durand, D.; Smith, Jeremy C.; Desmadril, Michel; Minard, Philippe; Douillard, Roger

doi:10.1051/jp4:1993849

Cited by 12 publications

(12 citation statements)

References 5 publications

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“…However, this range is known to be more limited for unfolded proteins, since the higher order terms in the expansion from which the Guinier approximation is derived are larger for more extended conformations 44,71 . Indeed, using increasing values of q max for Guinier fits leads to a systematic underestimation of R g , as seen in Figure 3b, where we have fitted the Guinier region for R17d in 4.96 M GdmCl to different ranges of

q_{\max} R_{g}

, clearly showing that the fitted radii of gyration become progressively smaller as

q_{\max} R_{g}

increases.…”

Section: Resultsmentioning

confidence: 99%

Consistent View of Polypeptide Chain Expansion in Chemical Denaturants from Multiple Experimental Methods

et al. 2016

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There has been a long-standing controversy regarding the effect of chemical denaturants on the dimensions of unfolded and intrinsically disordered proteins: A wide range of experimental techniques suggest that polypeptide chains expand with increasing denaturant concentration, but several studies using small-angle X-ray scattering (SAXS) reported no such increase of the radius of gyration (Rg). This inconsistency challenges our current understanding of the mechanism of chemical denaturants, which are widely employed to investigate protein folding and stability. Here, we use a combination of single-molecule Förster resonance energy transfer (FRET), SAXS, dynamic light scattering (DLS), and two-focus fluorescence correlation spectroscopy (2f-FCS) to characterize the denaturant dependence of the unfolded state of the spectrin domain R17 and the intrinsically disordered protein ACTR in two different denaturants. Standard analysis of the primary data clearly indicates an expansion of the unfolded state with increasing denaturant concentration irrespective of the protein, denaturant, or experimental method used. This is the first case in which SAXS and FRET have yielded even qualitatively consistent results regarding expansion in denaturant when applied to the same proteins. To more directly illustrate this self-consistency, we have used both SAXS and FRET data in a Bayesian procedure to refine structural ensembles representative of the observed unfolded state. This analysis demonstrates that both of these experimental probes are compatible with a common ensemble of protein configurations for each denaturant concentration. Furthermore, the resulting ensembles reproduce the trend of increasing hydrodynamic radius with denaturant concentration obtained by 2f-FCS and DLS. We were thus able to reconcile the results from all four experimental techniques quantitatively, to obtain a comprehensive structural picture of denaturant-induced unfolded state expansion, and to identify the most likely sources of earlier discrepancies.

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q_{\max} R_{g}

, clearly showing that the fitted radii of gyration become progressively smaller as

q_{\max} R_{g}

increases.…”

Section: Resultsmentioning

confidence: 99%

Consistent View of Polypeptide Chain Expansion in Chemical Denaturants from Multiple Experimental Methods

et al. 2016

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“…A direct experimental determination of R G for proteins unfolded by GuHCI has been achieved for only one of the proteins which were used to derive the scaling law mentioned above. Using small-angle neutron scattering on yeast phosphoglycerate kinase in 4 M GuHC1, Calmettes et al [18] obtained R G --7.8 nm from the Guinier approximation and R n = 9.2 nm from the Debye function for a random coil. We measured a Stokes' radius R~ = 5.66 nm.…”

Section: Discussionmentioning

confidence: 99%

Reduced‐denatured ribonuclease A is not in a compact state

et al. 1996

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Dynamic light scattering and circular dichroism experiments were performed to determine the compactness and residual secondary structure of reduced and by 6 M guanidine hydrochloride denatured ribonuclease A. We find that reduction of the four disulphide bonds by dithiothreitol at 20°C leads to total unfolding and that a temperature increase has no further effect on the dimension. The Stokes' radius of ribonuclease A at 20°C is R S = (1.90 4" 0.04) nm (native) and Rs = (3.14 4" 0.06) nm (reduced-denatured). Furthermore, circular dichroism spectra do not indicate any residual secondary structure. We suggest that reduced-denatured Ribonuclease A has a random coil-like conformation and is not in a compact denatured state.

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“…Samples for SANS experiments were prepared as detailed elsewhere 26. Protein concentrations, c , were varied from 3.75 to 20 mg mL −1 .…”

Section: Methodsmentioning

confidence: 99%

Study of structure and scaling behavior of chemically unfolded β‐casein by means of small‐angle neutron scattering

Aschi¹,

Gharbi²,

Daoud³

et al. 2006

Polymer International

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Structures of β‐casein formed in water with various concentrations of guanidine hydrochloride (GdmCl) were studied by small‐angle neutron scattering (SANS). A transition in structure and properties of the protein seems to occur around a GdmCl concentration of 1.2 mol L−1. The data are interpreted assuming that the protein molecules behave like multi‐block copolymers with alternating hydrophilic and hydrophobic sequences. Below the transition, the protein structure is still native and has a fractal dimension larger than that beyond the transition where the β‐casein has the feature of a three‐dimensional excluded‐volume coil. A possible interpretation of these results is that the presence of salt increases the value of the critical micelle concentration. This allows a comparison of the structure of β‐casein and synthetic macromolecules. We find that, although the protein is short, excluded‐volume interactions between monomers are present, and that the structure of β‐casein is very similar to what was found in dilute solutions of linear polymers in a good solvent. Thus, it is reasonable to assume that above the critical salt concentration, the protein is completely isolated, whereas below, some structure remains present. Copyright © 2006 Society of Chemical Industry

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Structure of proteins unfolded by guanidinium chloride

Abstract: Small-angle neutron scattering measurements were performed on two very different proteins unfolded by guanidinium chloride. It is shown that the neutron spectra given by unfolded phosphoglycerate kinase and β-casein are similar to those of excluded volume homopolymer chains

Cited by 12 publications

References 5 publications

Consistent View of Polypeptide Chain Expansion in Chemical Denaturants from Multiple Experimental Methods

Consistent View of Polypeptide Chain Expansion in Chemical Denaturants from Multiple Experimental Methods

Reduced‐denatured ribonuclease A is not in a compact state

Study of structure and scaling behavior of chemically unfolded β‐casein by means of small‐angle neutron scattering

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