Site-specific Dimensions Across a Highly Denatured Protein; A Single Molecule Study

McCarney, Evan R.; Werner, James H.; Bernstein, Seldon E.; Ruczinski, Ingo; Makarov, Dmitrii E.; Goodwin, Peter M.; Plaxco, Kevin W.

doi:10.1016/j.jmb.2005.07.015

Cited by 65 publications

(73 citation statements)

References 40 publications

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“…Its radius of gyration in 6 M GdmCl calculated from all labeled variants [using Eq. 4 and R g 2 ϭ ͗r 2 ͘/6 (21)] is 2.9 (Ϯ0.1) nm excluding dye linkers, in agreement with the 2.4 (ϩ0.4/Ϫ0.2) nm calculated from the scaling law given by Kohn et al (30,51). But how does the collapse of the unfolded state at low GdmCl concentrations affect the intramolecular distance distributions?…”

Section: Discussionsupporting

confidence: 84%

“…Identified bursts were corrected for background, differences in quantum yields, the different collection efficiencies of the detection channels, crosstalk, and direct acceptor excitation with the matrix approach (24) (SI Materials and Methods). For determining l p , the length of dyes and linkers were assumed to be equivalent to an additional 9 aa total, comparable with previous estimates (14,17,51,65). Fluorescence lifetime distribution analysis was performed as described by Laurence et al (17), except that the time-correlated single-photon counting histograms obtained from the unfolded state subpopulations were analyzed in terms of donor-acceptor distance distributions of a Gaussian chain (38,40) …”

Section: (For Details See Si Materials and Methods)mentioning

confidence: 99%

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Mapping protein collapse with single-molecule fluorescence and kinetic synchrotron radiation circular dichroism spectroscopy

Hoffmann

Kane

Nettels

et al. 2007

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

217

338

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We have used the combination of single-molecule Fö rster resonance energy transfer and kinetic synchrotron radiation circular dichroism experiments to probe the conformational ensemble of the collapsed unfolded state of the small cold shock protein CspTm under near-native conditions. This regime is physiologically most relevant but difficult to access experimentally, because the equilibrium signal in ensemble experiments is dominated by folded molecules. Here, we avoid this problem in two ways. One is the use of single-molecule Fö rster resonance energy transfer, which allows the separation of folded and unfolded subpopulations at equilibrium and provides information on long-range intramolecular distance distributions. From experiments with donor and acceptor chromophores placed at different positions within the chain, we find that the distance distributions in unfolded CspTm agree surprisingly well with a Gaussian chain not only at high concentrations of denaturant, where the polypeptide chain is expanded, but also at low denaturant concentrations, where the chain is collapsed. The second, complementary approach is synchrotron radiation circular dichroism spectroscopy of collapsed unfolded molecules transiently populated with a microfluidic device that enables rapid mixing. The results indicate a ␤-structure content of the collapsed unfolded state of Ϸ20% compared with the folded protein. This suggests that collapse can induce secondary structure in an unfolded state without interfering with long-range distance distributions characteristic of a random coil, which were previously found only for highly expanded unfolded proteins.Gaussian chain ͉ microfluidic mixing ͉ protein folding ͉ random coil ͉ secondary structure W ith the discovery of small proteins that fold in the absence of populated intermediates (1), our quantitative understanding of the elementary properties of protein folding reactions has made significant advances, including the structural characterization of transition states for folding (2) and the prediction of folding rates from native structure (3-5). One of the most severe limitations for the further development of these approaches is our ignorance about the energetic or structural properties of unfolded † † states of proteins. Because of the structural heterogeneity and complexity of the ensembles of conformations populated by unfolded proteins, their experimental characterization has proven extremely difficult. Traditional methods, such as small-angle scattering techniques (6), provide only global physical properties, e.g., the radius of gyration. In some cases, more detailed structural information can be obtained from NMR (7-10), but these studies usually provide information about the denatured state only under nonnative conditions, typically in the presence of large concentrations of denaturant, or through severe destabilization of the native state induced by covalent modification or mutations. The most interesting and physiologically relevant situation, however, is that of an unfolded sta...

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Section: Discussionsupporting

confidence: 84%

Section: (For Details See Si Materials and Methods)mentioning

confidence: 99%

Mapping protein collapse with single-molecule fluorescence and kinetic synchrotron radiation circular dichroism spectroscopy

Hoffmann

Kane

Nettels

et al. 2007

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

217

338

View full text Add to dashboard Cite

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“…(2), b = 0.38 nm is the distance between two C α -atoms, ν = 1 2 , l p = 0.4 nm is the persistence length of unfolded proteins in good solvent obtained from SAXS-results, 14,22 and N bonds = N + 9 takes into account the length of the fluorophore linker. 14,39 In order to relate the distribution of radii of gyration P(r G , ε, R G ) to a distance distribution P(r, ε, R G ), which is required to describe the mean transfer efficiencies E , we used the conditional probability function P(r|r G ), 13 which relates distance distribution of two random points inside a sphere with the radius of the sphere δ · r G :…”

Section: Calculation Of Radii Of Gyration From Mean Transfer Efficienmentioning

confidence: 99%

Single-molecule spectroscopy of the unexpected collapse of an unfolded protein at low pH

Hofmann

Nettels

Schuler

2013

The Journal of Chemical Physics

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The dimensions of intrinsically disordered and unfolded proteins critically depend on the solution conditions, such as temperature, pH, ionic strength, and osmolyte or denarurant concentration. However, a quantitative understanding of how the complex combination of chain-chain and chain-solvent interactions is affected by the solvent is still missing. Here, we take a step towards this goal by investigating the combined effect of pH and denaturants on the dimensions of an unfolded protein. We use single-molecule fluorescence spectroscopy to extract the dimensions of unfolded cold shock protein (CspTm) in mixtures of the denaturants urea and guanidinium chloride (GdmCl) at neutral and acidic pH. Surprisingly, even though a change in pH from 7 to 2.9 increases the net charge of CspTm from -3.8 to +10.2, the radius of gyration of the chain is very similar under both conditions, indicating that protonation of acidic side chains at low pH results in additional hydrophobic interactions. We use a simple shared binding site model that describes the joint effect of urea and GdmCl, together with polyampholyte theory and an ion cloud model that includes the chemical free energy of counterion interactions and side chain protonation, to quantify this effect. The dimensions of intrinsically disordered and unfolded proteins critically depend on the solution conditions, such as temperature, pH, ionic strength, and osmolyte or denarurant concentration. However, a quantitative understanding of how the complex combination of chain-chain and chain-solvent interactions is affected by the solvent is still missing. Here, we take a step towards this goal by investigating the combined effect of pH and denaturants on the dimensions of an unfolded protein. We use single-molecule fluorescence spectroscopy to extract the dimensions of unfolded cold shock protein (CspTm) in mixtures of the denaturants urea and guanidinium chloride (GdmCl) at neutral and acidic pH. Surprisingly, even though a change in pH from 7 to 2.9 increases the net charge of CspTm from −3.8 to +10.2, the radius of gyration of the chain is very similar under both conditions, indicating that protonation of acidic side chains at low pH results in additional hydrophobic interactions. We use a simple shared binding site model that describes the joint effect of urea and GdmCl, together with polyampholyte theory and an ion cloud model that includes the chemical free energy of counterion interactions and side chain protonation, to quantify this effect. © 2013 AIP Publishing LLC.

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“…This relationship can be tested, and the value of m estimated, by experimentally measuring distances between individual atom pairs within a protein, for instance by Förster resonance energy transfer (FRET). 22,23 Carrying out measurements of this type for a series of labeled proteins is a major undertaking, however, and the individual measurements may be overly sensitive to local structures or interactions with the fluorescent probes. An alternative approach to assess the internal scaling exponent is based on small-angle scattering by X-rays or neutrons (SANS).…”

Section: Introductionmentioning

confidence: 99%

Fractal dimension of an intrinsically disordered protein: Small‐angle X‐ray scattering and computational study of the bacteriophage λ N protein

2011

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Small-angle X-ray scattering (SAXS) was used to characterize the bacteriophage k N protein, a 107 residue intrinsically disordered protein (IDP) that functions as a transcriptional antitermination factor. The SAXS data were used to estimate both the average radius of gyration and the fractal dimension, a measure of the protein's internal scaling properties, under a variety of solution conditions. In the absence of denaturants, the radius of gyration was 38 6 3.5 Å and the fractal dimension was 1.76 6 0.05, slightly larger than the value predicted for a well-solvated polymer with excluded volume (1.7). Neither the radius of gyration nor the fractal dimension changed significantly on the addition of urea, further indicating that the protein is extensively unfolded and well solvated in the absence of denaturant. The addition of NaCl or D 2 O was found to promote aggregation, but did not appear to affect the properties of the monomeric form. The experimental SAXS profiles were also compared with those predicted by a computational model for a random-coil polypeptide, with an adjustable solvation energy term. The experimental data were well fit to the model with the solvation energy close to zero. These results indicate that the k N protein is among the more expanded members of the broad class of IDPs, most likely because of its high content of charged residues and a large net charge (115 at neutral pH). The expanded nature of the conformational ensemble may play a role in facilitating the interactions of the protein with other components of the dynamic transcriptional complex.

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Site-specific Dimensions Across a Highly Denatured Protein; A Single Molecule Study

Cited by 65 publications

References 40 publications

Mapping protein collapse with single-molecule fluorescence and kinetic synchrotron radiation circular dichroism spectroscopy

Mapping protein collapse with single-molecule fluorescence and kinetic synchrotron radiation circular dichroism spectroscopy

Single-molecule spectroscopy of the unexpected collapse of an unfolded protein at low pH

Fractal dimension of an intrinsically disordered protein: Small‐angle X‐ray scattering and computational study of the bacteriophage λ N protein

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