Use of high-speed size-exclusion chromatography for the study of protein folding and stability

Corbett, Ronald J. T.; Roche, Rodney S.

doi:10.1021/bi00303a047

Cited by 183 publications

(138 citation statements)

References 28 publications

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“…of U ~ N transition) can be as large as ~ 40 min at 23°C [1,2], which suggests the possibility of observing a bimodal equilibrium size distribution even at room temperature. This distribution has been actually observed for equilibrium urea-induced unfolding of myoglobin [18] and of bovine serum albumin [19] at 25°C. The U -> MG transition usually takes place in a few seconds [4,7].…”

Section: Resultssupporting

confidence: 67%

‘All‐or‐none’ mechanism of the molten globule unfolding

et al. 1992

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The Gdm-HCl-induced unfolding of bovine carbonic anhydrase B and S. at~ret~s fl-lactamase was studied at 4°C by a variety of methods. With the use of FPLC it has been shown that within the transition from the molten globule to the unfolded state the distribution function of molecular dimensions is bimodal. This means that equilibrium intermediates between the molten globule and the unfolded states are about, i.¢. the molten globule unfolding follows the "all-or-none' mechanism.

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Section: Resultssupporting

confidence: 67%

‘All‐or‐none’ mechanism of the molten globule unfolding

et al. 1992

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“…The R S values allow a direct comparison of analyte "size", without being obscured by the different solvent viscosities of the individual experiments [74]. In solutions of 0 and 30% acetonitrile content at pH 10, the R S values of heme in Mb solution are close to those of the protein, which reflects the fact that the two species form a stable noncovalent complex.…”

Section: Stokes Radiimentioning

confidence: 96%

“…This notion is compatible with the data presented here. The Stokes radius is a measure of the volume occupied by a protein [74], and there is a qualitative correlation between the measured R S values, and the corresponding shifts seen in the ESI charge state distribution of the protein.…”

Section: Stokes Radiimentioning

confidence: 99%

Diffusion measurements by electrospray mass spectrometry for studying solution-phase noncovalent interactions

Clark

Konermann

2003

J. Am. Soc. Mass Spectrom.

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This study describes a novel approach for monitoring noncovalent interactions in solution by electrospray mass spectrometry (ESI-MS). The technique is based on measurements of analyte diffusion in solution. Diffusion coefficients of a target macromolecule and a potential low molecular weight binding partner are determined by measuring the spread of an initially sharp boundary between two solutions of different concentration in a laminar flow tube (Taylor dispersion), as described in Rapid Commun. Mass Spectrom. 2002Spectrom. , 16, 1454Spectrom. -1462. In the absence of noncovalent interactions, the measured ESI-MS dispersion profiles are expected to show a gradual transition for the macromolecule and a steep transition for the low molecular weight compound. However, if the two analytes form a noncovalent complex in solution the dispersion profiles of the two species will be very similar, since the translational diffusion of the small compound is determined by the slow Brownian motion of the macromolecule. In contrast to conventional ESI-MS-based techniques for studying noncovalent complexes, this approach does not rely on the preservation of solution-phase interactions in the gas phase. On the contrary, "harsh" conditions at the ion source are required to disrupt any potential gasphase interactions between the two species, such that their dispersion profiles can be monitored separately. The viability of this technique is demonstrated in studies on noncovalent heme-protein interactions in myoglobin. Tight noncovalent binding is observed in solutions of pH 10, both in the absence and in the presence of 30% acetonitrile. In contrast, a significant disruption of the noncovalent interactions is seen at an acetonitrile content of 50%. Under these conditions, the diffusion coefficient of heme in the presence of myoglobin is only slightly lower than that of heme in a protein-free solution. A breakdown of the noncovalent interactions is also observed in aqueous solution of pH 2.4, where myoglobin is known to adopt an acid-unfolded conformation. (J Am Soc Mass Spectrom 2003, 14, 430 -441)

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“…The Stokes radius was determined using the procedure of Corbett and Roche (1984). Protein sample concentrations were 2 pM.…”

Section: A L Fink Et Aimentioning

confidence: 99%

Characterization of the stable, acid‐induced, molten globule‐like state of staphylococcal nuclease

et al. 1993

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Titration of a salt-free solution of native staphylococcal nuclease by HCl leads to an unfolding transition in the vicinity of pH 4, as determined by near-and far-UV circular dichroism. At pH 2-3, the protein is substantially unfolded. The addition of further HCI results in a second transition, this one to a more structured species (the A state) with the properties of an expanded molten globule, namely substantial secondary structure, little or no tertiary structure, relatively compact size as determined by hydrodynamic radius, and the ability to bind the hydrophobic dye I-anilino-%naphthalene sulfonic acid. The addition of anions, in the form of neutral salts, to the acid-unfolded state at pH 2 also causes a transition leading to the A state. Fourier transform infrared analysis of the amide I band was used to compare the amount and type of secondary structure in the native and A states.A significant decrease in a-helix structure, with a corresponding increase in 6 or extended structure, was observed in the A state, compared to the native state. A model to account for such compact denatured states is proposed.

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Use of high-speed size-exclusion chromatography for the study of protein folding and stability

Cited by 183 publications

References 28 publications

‘All‐or‐none’ mechanism of the molten globule unfolding

‘All‐or‐none’ mechanism of the molten globule unfolding

Diffusion measurements by electrospray mass spectrometry for studying solution-phase noncovalent interactions

Characterization of the stable, acid‐induced, molten globule‐like state of staphylococcal nuclease

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