Boundary analysis of sedimentation‐velocity experiments with monodisperse and paucidisperse solutes

Holde, Kensal E. van; Weischet, Wolfgang

doi:10.1002/bip.1978.360170602

Cited by 324 publications

(294 citation statements)

References 5 publications

(1 reference statement)

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“…Absorbance scans were collected at 50,000 rpm. The sedimentation velocity data were analyzed by the method of van Holde and Weischet (28). The sedimentation coefficients, s 20,w were calculated using the UltraScan (version 6.2.0.)…”

Section: Methodsmentioning

confidence: 99%

Irreversible Thermal Denaturation of Glucose Oxidase from Aspergillus niger Is the Transition to the Denatured State with Residual Structure

Žoldák

Zubrik

Musatov

et al. 2004

Journal of Biological Chemistry

127

105

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Glucose oxidase (GOX; ␤-D-glucose:oxygen oxidoreductase) from Aspergillus niger is a dimeric flavoprotein with a molecular mass of 80 kDa/monomer. Thermal denaturation of glucose oxidase has been studied by absorbance, circular dichroism spectroscopy, viscosimetry, and differential scanning calorimetry. Thermal transition of this homodimeric enzyme is irreversible and, surprisingly, independent of GOX concentration (0.2-5.1 mg/ml). It has an apparent transition temperature of 55.8 ؎ 1.2°C and an activation energy of ϳ280 kJ/mol, calculated from the Lumry-Eyring model. The thermally denatured state of GOX after recooling has the following characteristics. (i) It retains ϳ70% of the native secondary structure ellipticity; (ii) it has a relatively low intrinsic viscosity, 7.5 ml/g; (iii) it binds ANS; (iv) it has a low Stern-Volmer constant of tryptophan quenching; and (v) it forms defined oligomeric (dimers, trimers, tetramers) structures. It is significantly different from chemically denatured (6.67 M GdmHCl) GOX. Both the thermal and the chemical denaturation of GOX cause dissociation of the flavin cofactor; however, only the chemical denaturation is accompanied by dissociation of the homodimeric GOX into monomers. The transition temperature is independent of the protein concentration, and the properties of the thermally denatured protein indicate that thermally denatured GOX is a compact structure, a form of molten globulelike apoenzyme. GOX is thus an exceptional example of a relatively unstable mesophilic dimeric enzyme with residual structure in its thermally denatured state.Glucose oxidase (GOX 1 ; ␤-D-glucose:oxygen oxidoreductase, EC 1.1.3.4) is a flavoenzyme that catalyzes oxidation of ␤-Dglucose by molecular oxygen to ␦-gluconolactone, which subsequently hydrolyzes spontaneously to gluconic acid and hydrogen peroxide. The enzyme contains one tightly, noncovalently bound FAD cofactor per monomer and is a homodimer with a molecular mass of 160 kDa, depending on the extent of glycosylation (1). Glucose oxidase from Aspergillus niger is glycosylated by neutral sugars (mostly mannose-like sugars) and by amino sugars (2). Several reports find that the sugar content may vary from 11 up to 30% (3-5).GOX is of considerable commercial importance. The enzyme has applications in the food and fermentation industry, in the textile industry, and as a molecular diagnostic and analytical tool in medical and environmental monitoring applications (6 -10).The study of GOX and its applications is limited by its conformational instability. A significant effort has been made to increase the stability of GOX. It is known that both the thermal stability and the dynamic properties of the enzyme depend on its redox state (11, 12). Protein glycosylation affects the conformational dynamics of the active site and thus the activity of the enzyme (13). However, the way in which glycosylation affects the stability of GOX is not known. On the other hand, modification of the glucose oxidase surface by artificial long polyethylene-glycol ...

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Section: Methodsmentioning

confidence: 99%

Irreversible Thermal Denaturation of Glucose Oxidase from Aspergillus niger Is the Transition to the Denatured State with Residual Structure

Žoldák

Zubrik

Musatov

et al. 2004

Journal of Biological Chemistry

127

105

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“…Solute distributions were recorded at 7.5-min intervals with a radial step size of 0.001 cm using scanning absorbance optics at a wavelength of 280 nm. The data were analyzed using the Van Holde-Weischet analysis method (26), which is implemented in the UltraScan II software package (Version 5.0 for Unix) of B. Demeler.…”

Section: Methodsmentioning

confidence: 99%

A Novel, Enzymatically Active Conformation of the Escherichia coli NADH:Ubiquinone Oxidoreductase (Complex I)

Böttcher

Scheide

Hesterberg

et al. 2002

Journal of Biological Chemistry

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Electron microscopy has demonstrated the unusual L-shaped structure of the respiratory complex I consisting of two arms, which are arranged perpendicular to each other. We found that the Escherichia coli complex I has an additional stable conformation, with the two arms arranged side by side, resulting in a horseshoeshaped structure. The structure of both conformations was determined by means of electron microscopy of gold thioglucose-stained single particles. They were distinguished from each other by titration of the complex with polyethylene glycol and by means of analytical ultracentrifugation. The transition between the two conformations is induced by the ionic strength of the buffer and is reversible. Only the horseshoe-shaped complex I exhibits enzyme activity in detergent solution, which is abolished by the addition of salt. Therefore, it is proposed that this structure is the native conformation of the complex in the membrane.

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“…For complicated systems containing multiple species we used the van Holde and Weischet global boundary analysis for fitting the absorbance data (42). The van Holde and Weischet global fitting method is very useful for examining sample homogeneity/heterogeneity and facilitates a rigorous analysis of complex boundaries of multicomponent systems, yielding accurate S 20,w for all components present (43).…”

Section: Methodsmentioning

confidence: 99%

Characterization of the Transthyretin Acid Denaturation Pathways by Analytical Ultracentrifugation: Implications for Wild-Type, V30M, and L55P Amyloid Fibril Formation

1998

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Analytical ultracentrifugation methods were utilized to further characterize the acid denaturation pathways of wild-type, V30M, and L55P transthyretin (TTR) that generate intermediates leading to amyloid fibril formation and possibly the diseases senile systemic amyloidosis and familial amyloid polyneuropathy. Equilibrium and velocity methods were employed herein to characterize the TTR quaternary structural requirements for amyloid fibril formation. From neutral to slightly acidic conditions (pH 7.5-5.1), wildtype transthyretin (0.2-0.3 mg/mL, 100 mM KCl, 37°C) exists as a tetramer and is incapable of fibril formation. Under more acidic conditions (pH 5 to 3.9), tetrameric wild-type TTR slowly dissociates to a monomer having an alternatively folded tertiary structure(s) that self-assembles at physiological concentration (0.2 mg/mL) into a ladder of quaternary structural intermediates of increasing molecular weight. These intermediates appear to be on the pathway of amyloid fibril formation, since they ultimately disappear when amyloid fibrils are observed. The V30M and L55P TTR variants exhibit similar acid denaturation pathways, with the exception that dissociation of the tetramer to the monomeric amyloidogenic intermediate occurs at a higher pH and to a much greater extent, allowing the quaternary structural intermediates to be readily observed by velocity methods. Partial denaturation and assembly of the monomeric amyloidogenic intermediate(s) occur at pH 5.4 for V30M and L55P TTR over a 72 h period, during which wild-type TTR maintains its normal tetrameric three-dimensional structure. Interestingly, the L55P and V30M familial amyloid polyneuropathy (FAP) associated variants form amyloid protofilaments at pH 7.5 (37°C) after several weeks of incubation, suggesting that the activation barriers for TTR tetramer dissociation to the monomeric amyloidogenic intermediate are much lower for the FAP variants relative to wild-type TTR, which does not form amyloid or amyloid protofilaments under these conditions. This study establishes the key role of the monomeric amyloidogenic intermediate and its selfassembly into a ladder of quaternary structural intermediates for the formation of wild-type, V30M, and L55P transthyretin amyloid fibrils.

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Boundary analysis of sedimentation‐velocity experiments with monodisperse and paucidisperse solutes

Cited by 324 publications

References 5 publications

Irreversible Thermal Denaturation of Glucose Oxidase from Aspergillus niger Is the Transition to the Denatured State with Residual Structure

Irreversible Thermal Denaturation of Glucose Oxidase from Aspergillus niger Is the Transition to the Denatured State with Residual Structure

A Novel, Enzymatically Active Conformation of the Escherichia coli NADH:Ubiquinone Oxidoreductase (Complex I)

Characterization of the Transthyretin Acid Denaturation Pathways by Analytical Ultracentrifugation: Implications for Wild-Type, V30M, and L55P Amyloid Fibril Formation

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