Cold Denaturation of Barstar:1H,15N and13C NMR Assignment and Characterisation of Residual Structure

Wong, Kam‐Bo; Freund, Stefan; Fersht, Alan R.

doi:10.1006/jmbi.1996.0359

Cited by 104 publications

(108 citation statements)

References 61 publications

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“…13 However, spectra recorded at low and high temperatures are very different: the spectrum at − 1°C is well populated and informative, even though most of the nuclear Overhauser enhancement effects are intra-residue or sequential as previously reported. 8 The 50°C nuclear Overhauser enhancement spectroscopy-HSQC spectrum contains instead very few off-diagonal peaks (Fig.…”

Section: Resultsmentioning

confidence: 69%

“…Ethylenediaminetetraacetic acid and salts were removed by dialysis prior to concentration of the protein. 15 Nlabeled and 15 N, 13 C-double-labeled samples were produced by growing the bacteria in minimal medium using labeled ammonium sulfate and glucose as the sole source of nitrogen and carbon.…”

Section: Experimental Procedures Sample Preparationmentioning

confidence: 99%

“…15 N-and 15 N, 13 C-labeled Yhf1 samples used for NMR studies (ca 0.5 mM) were dialyzed to 20 mM Hepes at pH 7.0 and 2 mM DTT, containing 10% (v/v) D 2 O. All experiments were carried out on a Bruker Avance spectrometer operating at 16.4 T and equipped with an inverse triple-resonance single-axis gradient cryoprobe.…”

Section: Nmr Spectroscopymentioning

confidence: 99%

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The Role of Hydration in Protein Stability: Comparison of the Cold and Heat Unfolded States of Yfh1

Adrover

Martorell

Martin³

et al. 2012

Journal of Molecular Biology

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Protein unfolding occurs at both low and high temperatures, although in most cases, only the high-temperature transition can be experimentally studied. A pressing question is how much the low-and high-temperature denatured states, although thermodynamically equivalent, are structurally and kinetically similar. We have combined experimental and computational approaches to compare the high-and low-temperature unfolded states of Yfh1, a natural protein that, at physiologic pH, undergoes cold and heat denaturation around 0°C and 40°C without the help of ad hoc destabilization. We observe that the two denatured states have similar but not identical residual secondary structures, different kinetics and compactness and a remarkably different degree of hydration. We use molecular dynamics simulations to rationalize the role of solvation and its effect on protein stability.Crown

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Section: Resultsmentioning

confidence: 69%

Section: Experimental Procedures Sample Preparationmentioning

confidence: 99%

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The Role of Hydration in Protein Stability: Comparison of the Cold and Heat Unfolded States of Yfh1

Adrover

Martorell

Martin³

et al. 2012

Journal of Molecular Biology

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“…Recent experimental data suggest that unfolded states of proteins are ensembles of rapidly interconverting, structurally and dynamically diverse conformers that may be fairly compact and exhibit considerable amounts of nonrandom structure. Residual secondary and tertiary structure can be retained in unfolded states, involving specific local and longrange interactions, some of which are native-like and some of which are not observed in the native (folded) structure (1)(2)(3)(4)(5).…”

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confidence: 99%

Site-specific contributions to the pH dependence of protein stability

Tollinger

Crowhurst

Kay

et al. 2003

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

126

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Understanding protein stability is a significant challenge requiring characterization of interactions within both folded and unfolded states. Of these, electrostatic interactions influence ionization equilibria of acidic and basic groups and diversify their pK a values. The pH dependence of the thermodynamic stability (⌬G FU) of a protein arises as a consequence of differential pK a values between folded and unfolded states. Previous attempts to calculate pHdependent contributions to stability have been limited by the lack of experimental unfolded state pK a values. Using recently developed NMR spectroscopic methods, we have determined residuespecific pK a values for a thermodynamically unstable Src homology 3 domain in both states, enabling the calculation of the pH dependence of stability based on simple analytical expressions. The calculated pH stability profile obtained agrees very well with experiment, unlike profiles derived from two current models of electrostatic interactions within unfolded states. Most importantly, per-residue contributions to the pH dependence of ⌬G FU derived from the data provide insights into specific electrostatic interactions in both the folded and unfolded states and their roles in protein stability. These interactions include a hydrogen bond between the Asp-8 side-chain and the Lys-21 backbone amide group in the folded state, which represents a highly conserved interaction in Src homology 3 domains.

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“…21 Other experiments, studying urea-unfolded Barstar, also find residual structure within the unfolded conformations. For example, five residues in Helix 1 show propensity towards populating helical regions in (φ,ψ) space.…”

Section: This Studymentioning

confidence: 97%

A study of Barstar folding events using boundary value simulations

Yunger

2007

Physica A: Statistical Mechanics and its Applications

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This study revolves around a computational algorithm called SDEL (Stochastic Difference Equation in Length) that generates approximate protein folding trajectories on the atomically detailed resolution scale. The protein studied is Barstar-a barnase inhibitor. Because of the protein's interesting structure (four alpha helices, three beta strands) and relatively small size (89 residues), Barstar is an optimal choice for running complete folding trajectories on a computer. 12 pathways were generated with SDEL, starting from a structurally wide selection of unfolded conformations, yet all ending with the native configuration. We tracked hydrogen bonds, dihedral angles, native and non-native contacts, and energetic along these folding pathways. The resulting trajectories show: 1) Barstar follows the Hydrophobic Collapse folding scenario, 2) native α-helices begin forming earlier in the trajectory than the β-sheets, 3) particular residues maintain a propensity for helical structure in their unfolded state, and 4) specific non-native contacts persist during the folding trajectory. Strong correlations were found between the SDEL pathways and data from NMR, CD, and other experimental studies.

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Cold Denaturation of Barstar:1H,15N and13C NMR Assignment and Characterisation of Residual Structure

Cited by 104 publications

References 61 publications

The Role of Hydration in Protein Stability: Comparison of the Cold and Heat Unfolded States of Yfh1

The Role of Hydration in Protein Stability: Comparison of the Cold and Heat Unfolded States of Yfh1

Site-specific contributions to the pH dependence of protein stability

A study of Barstar folding events using boundary value simulations

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