Unfolding and Conformational Distributions during Protein Precipitation

Chang, Stephen T.; Tobler, Scott A.; Fernandez, Erik J.

doi:10.1021/bp010025h

Cited by 7 publications

(7 citation statements)

References 12 publications

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“…At one level, this is a demonstration of stability in the adsorbed state-the adsorption of the protein on the surface has not slowed the overall observed refolding reaction enough to change the exchange kinetics from an EX2 to EX1 regime. This is in contrast to our prior observations of such a switch during protein precipitation under destabilizing (Chang et al, 2001) and aggregation (Tobler et al, 2000). In those prior studies, the addition of a protein self-association process under denaturing conditions trapped protein molecules in an unfolded form, reducing the overall rate of refolding.…”

Section: Discussion Lysozyme Hydrogen Exchange Measurementscontrasting

confidence: 99%

Hydrophobic interaction chromatography selectivity changes among three stable proteins: conformation does not play a major role

Jones¹,

Fernandez²

2004

Biotech & Bioengineering

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Interesting retention and selectivity changes have been noted for a number of proteins in hydrophobic interaction chromatography (HIC). In this study, we investigated the degree to which conformational changes may be responsible for selectivity changes of stable proteins. Hydrogen-deuterium isotope exchange detected by mass spectrometry was used to investigate changes in solvent accessibility during adsorption on HIC media. Lysozyme was determined to exhibit EX2 hydrogen exchange kinetics both in solution and adsorbed to Butyl Sepharose 4 Fast Flow and Phenyl Sepharose 6 Fast Flow high sub surfaces. A small, but significant, increase in solvent accessibility was observed upon adsorption. Similar approaches were used to analyze solvent accessibility of three stable proteins with melting temperatures above 50jC exhibiting significant selectivity changes on Butyl Sepharose and Toyopearl Butyl 650M. While all three proteins (lysozyme, chymotrypsinogen A, and ovalbumin) exhibited enhanced exchange while adsorbed, no differences in solvent accessibility on the different adsorbents were observed. More detailed studies of lysozyme showed no significant changes in labeling prior or during elution. These results demonstrate that HIC surfaces examined here do not dramatically alter the structure of these stable proteins and that differences in conformation are not responsible for the selectivity changes observed. Thus, other factors such as different preferred binding orientations or variations between the media pore structure, size, and/or surface chemistry must be responsible. B 2004 Wiley Periodicals, Inc.

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Section: Discussion Lysozyme Hydrogen Exchange Measurementscontrasting

confidence: 99%

Hydrophobic interaction chromatography selectivity changes among three stable proteins: conformation does not play a major role

Jones¹,

Fernandez²

2004

Biotech & Bioengineering

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“…Heat-Induced Inactivation of Lysozyme in the Presence of Additives. Lysozyme (pI ) 11) is commonly used as a model protein for misfolding and refolding (11,16,(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31). Figure 1 shows the residual activity of lysozyme after incubation at 98°C for 30 min.…”

Section: Resultsmentioning

confidence: 99%

Amino Acid Esters Prevent Thermal Inactivation and Aggregation of Lysozyme

Shiraki

Kudou

Sakamoto

et al. 2008

Biotechnol Progress

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Small potent inhibitors of aggregation are eagerly demanded for preventing the inactivation of proteins. This paper shows that amino acid esters (AAEs) prevent heat-induced aggregation and inactivation of hen egg lysozyme. Lysozyme was completely inactivated (<1% original activity) during heat treatment at 98 degrees C for 30 min in a solution containing 0.2 mg/mL lysozyme in 50 mM Na-phosphate buffer (pH 6.5). The residual activities only slightly increased (<5%) in the presence of 100 mM commonly used additives such as arginine, guanidine, urea, and sugars. However, in the presence of 100 mM AAEs, the residual activities were >60% and no aggregates were observed during the heat treatment at 98 degrees C for 30 min. This fact provides new information on the scaffold for designing additives to prevent heat-induced aggregation.

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“…Recent research on aggregation of lysozyme in aqueous solution reveals selective unfolding of the β-domains of the protein in these precipitates. 45 The interplay between microemulsion structure and protein conformation will be discussed in detail below.…”

Section: Resultsmentioning

confidence: 99%

“…The protein in this precipitate probably experiences conformational changes in select regions of the molecule and proteins are therefore not completely unfolded. Recent research on aggregation of lysozyme in aqueous solution reveals selective unfolding of the β-domains of the protein in these precipitates . The interplay between microemulsion structure and protein conformation will be discussed in detail below.…”

Section: Resultsmentioning

confidence: 99%

Changes in Microemulsion and Protein Structure in IgG−AOT−Brine−Isooctane Systems

Gerhardt

Dungan

2004

J. Phys. Chem. B

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Changes in structure of protein molecules and water-in-oil (w/o) microemulsion aggregates were investigated using the large protein immunoglobulin G (IgG, MW 155,000) and an equivolume oil/water mixture composed of brine, sulfosuccinic acid bis [2-ethylhexyl]ester (sodium salt) (AOT), and isooctane. The protein solution in the microemulsion phase was metastable: over time this solution changed, as protein and w/o droplets aggregated and precipitated to the interface between aqueous and organic phases. Such factors as AOT concentration, temperature, and salt concentration were found to influence the protein and surfactant structures in the microemulsion. Protein conformation was probed using circular dichroism spectroscopy whereas the microemulsion structure was determined from dynamic light scattering measurements. Protein conformation and microemulsion structure were found to have significant effects on protein stability in the microemulsion. The stabilizing effects of clusters formed at higher salt and/or AOT concentrations are discussed. IgG adopts an intermediate denatured state in the microemulsion phase close to the alternatively folded state known as the A state, with well-defined contacts in the tertiary structure immediately after phase equilibration. The change in protein conformation with time accompanied by the cluster growth eventually leads to the protein and surfactant transfer into a third, solid middle phase from the organic solution.

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Unfolding and Conformational Distributions during Protein Precipitation

Cited by 7 publications

References 12 publications

Hydrophobic interaction chromatography selectivity changes among three stable proteins: conformation does not play a major role

Hydrophobic interaction chromatography selectivity changes among three stable proteins: conformation does not play a major role

Amino Acid Esters Prevent Thermal Inactivation and Aggregation of Lysozyme

Changes in Microemulsion and Protein Structure in IgG−AOT−Brine−Isooctane Systems

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