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Effect of metal catalyzed oxidation in recombinant viral protein assemblies.

Castro-Acosta RM, Rodríguez-Limas WA, Valderrama B, Ramírez OT, Palomares LA - Microb. Cell Fact. (2014)

Bottom Line: Despite its importance, very few studies have investigated the effect of oxidation in protein assemblies and their structural units.It was found that assembly protected VP6 from in vitro metal-catalyzed oxidation.The in vitro assembly efficiency of VP6U into VP6NT decreased as the oxidant concentration increased.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, A,P, 510-3, C,P, 62210, Cuernavaca, Morelos, Mexico. laura@ibt.unam.mx.

ABSTRACT

Background: Protein assemblies, such as virus-like particles, have increasing importance as vaccines, delivery vehicles and nanomaterials. However, their use requires stable assemblies. An important cause of loss of stability in proteins is oxidation, which can occur during their production, purification and storage. Despite its importance, very few studies have investigated the effect of oxidation in protein assemblies and their structural units. In this work, we investigated the role of in vitro oxidation in the assembly and stability of rotavirus VP6, a polymorphic protein.

Results: The susceptibility to oxidation of VP6 assembled into nanotubes (VP6NT) and unassembled VP6 (VP6U) was determined and compared to bovine serum albumin (BSA) as control. VP6 was more resistant to oxidation than BSA, as determined by measuring protein degradation and carbonyl content. It was found that assembly protected VP6 from in vitro metal-catalyzed oxidation. Oxidation provoked protein aggregation and VP6NT fragmentation, as evidenced by dynamic light scattering and transmission electron microscopy. Oxidative damage of VP6 correlated with a decrease of its center of fluorescence spectral mass. The in vitro assembly efficiency of VP6U into VP6NT decreased as the oxidant concentration increased.

Conclusions: Oxidation caused carbonylation, quenching, and destruction of aromatic amino acids and aggregation of VP6 in its assembled and unassembled forms. Such modifications affected protein functionality, including its ability to assemble. That assembly protected VP6 from oxidation shows that exposure of susceptible amino acids to the solvent increases their damage, and therefore the protein surface area that is exposed to the solvent is determinant of its susceptibility to oxidation. The inability of oxidized VP6 to assemble into nanotubes highlights the importance of avoiding this modification during the production of proteins that self-assemble. This is the first time that the role of oxidation in protein assembly is studied, evidencing that oxidation should be minimized during the production process if VP6 nanotubes are required.

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Densitometric analysis of reducing 12% SDS-PAGE gels loaded with 2.5 µg of VP6NT, VP6U and BSA previously exposed to different oxidative treatments and stained with Coomassie blue. A) Exposure of protein samples to different concentrations of H2O2 for 6 h. B) Exposure of protein samples to metal catalyzed oxidation (MCO) with 150 µM of FeCl2 at various H2O2 concentrations for 1 h. C) Exposure of protein samples to MCO with 150 µM of FeCl2 at various H2O2 concentrations for 6 h. Measurements were performed to identically treated samples from triplicate experiments. Error bars represent standard deviations among experiments.
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Figure 2: Densitometric analysis of reducing 12% SDS-PAGE gels loaded with 2.5 µg of VP6NT, VP6U and BSA previously exposed to different oxidative treatments and stained with Coomassie blue. A) Exposure of protein samples to different concentrations of H2O2 for 6 h. B) Exposure of protein samples to metal catalyzed oxidation (MCO) with 150 µM of FeCl2 at various H2O2 concentrations for 1 h. C) Exposure of protein samples to MCO with 150 µM of FeCl2 at various H2O2 concentrations for 6 h. Measurements were performed to identically treated samples from triplicate experiments. Error bars represent standard deviations among experiments.

Mentions: Protein oxidation can result in degradation by fragmentation of the backbone, which can be evidenced by the disappearance of a stainable band in SDS-PAGE gels [15,17]. Degradation analysis was used to evaluate the susceptibility of nanotubes and disassembled VP6 to H2O2. For comparison, bovine serum albumin (BSA), a widely studied protein, was also subjected to oxidation. Gels were scanned and the intensity and area of each band were quantified by densitometry. Results are shown in Figure 2. Exposure to up to 10,000 µM H2O2 did not cause band disappearance in gels of treated BSA, VP6NT or VP6U, even after 6 h of incubation with the oxidant (Figure 2A). As VP6 was not degraded by exposition to H2O2, all following experiments were performed only with MCO. In contrast, when exposed to H2O2 in MCO, the VP6 and BSA bands disappeared although with different behavior (Figures 2B and C). While VP6, in either of its forms, resisted MCO up to 5 mM of H2O2 for 1 h, the BSA band decreased at H2O2 concentrations above 0.25 mM. Exposition to H2O2 in MCO for 6 h caused degradation of BSA at all concentrations tested, evidencing that it is less resistant to degradation than VP6. These experiments also showed that VP6NT are more resistant to oxidation than VP6U. While the VP6U band disappeared after exposure to 10,000 µM of H2O2 in MCO for 1 h, no change was observed in VP6NT when incubated under the same condition. Exposure of VP6NT to high H2O2 concentrations for as much as six hours was needed for its band to disappear, suggesting a higher stability towards oxidative insults. The same behavior was observed in native gels (data not shown).


Effect of metal catalyzed oxidation in recombinant viral protein assemblies.

Castro-Acosta RM, Rodríguez-Limas WA, Valderrama B, Ramírez OT, Palomares LA - Microb. Cell Fact. (2014)

Densitometric analysis of reducing 12% SDS-PAGE gels loaded with 2.5 µg of VP6NT, VP6U and BSA previously exposed to different oxidative treatments and stained with Coomassie blue. A) Exposure of protein samples to different concentrations of H2O2 for 6 h. B) Exposure of protein samples to metal catalyzed oxidation (MCO) with 150 µM of FeCl2 at various H2O2 concentrations for 1 h. C) Exposure of protein samples to MCO with 150 µM of FeCl2 at various H2O2 concentrations for 6 h. Measurements were performed to identically treated samples from triplicate experiments. Error bars represent standard deviations among experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3928578&req=5

Figure 2: Densitometric analysis of reducing 12% SDS-PAGE gels loaded with 2.5 µg of VP6NT, VP6U and BSA previously exposed to different oxidative treatments and stained with Coomassie blue. A) Exposure of protein samples to different concentrations of H2O2 for 6 h. B) Exposure of protein samples to metal catalyzed oxidation (MCO) with 150 µM of FeCl2 at various H2O2 concentrations for 1 h. C) Exposure of protein samples to MCO with 150 µM of FeCl2 at various H2O2 concentrations for 6 h. Measurements were performed to identically treated samples from triplicate experiments. Error bars represent standard deviations among experiments.
Mentions: Protein oxidation can result in degradation by fragmentation of the backbone, which can be evidenced by the disappearance of a stainable band in SDS-PAGE gels [15,17]. Degradation analysis was used to evaluate the susceptibility of nanotubes and disassembled VP6 to H2O2. For comparison, bovine serum albumin (BSA), a widely studied protein, was also subjected to oxidation. Gels were scanned and the intensity and area of each band were quantified by densitometry. Results are shown in Figure 2. Exposure to up to 10,000 µM H2O2 did not cause band disappearance in gels of treated BSA, VP6NT or VP6U, even after 6 h of incubation with the oxidant (Figure 2A). As VP6 was not degraded by exposition to H2O2, all following experiments were performed only with MCO. In contrast, when exposed to H2O2 in MCO, the VP6 and BSA bands disappeared although with different behavior (Figures 2B and C). While VP6, in either of its forms, resisted MCO up to 5 mM of H2O2 for 1 h, the BSA band decreased at H2O2 concentrations above 0.25 mM. Exposition to H2O2 in MCO for 6 h caused degradation of BSA at all concentrations tested, evidencing that it is less resistant to degradation than VP6. These experiments also showed that VP6NT are more resistant to oxidation than VP6U. While the VP6U band disappeared after exposure to 10,000 µM of H2O2 in MCO for 1 h, no change was observed in VP6NT when incubated under the same condition. Exposure of VP6NT to high H2O2 concentrations for as much as six hours was needed for its band to disappear, suggesting a higher stability towards oxidative insults. The same behavior was observed in native gels (data not shown).

Bottom Line: Despite its importance, very few studies have investigated the effect of oxidation in protein assemblies and their structural units.It was found that assembly protected VP6 from in vitro metal-catalyzed oxidation.The in vitro assembly efficiency of VP6U into VP6NT decreased as the oxidant concentration increased.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, A,P, 510-3, C,P, 62210, Cuernavaca, Morelos, Mexico. laura@ibt.unam.mx.

ABSTRACT

Background: Protein assemblies, such as virus-like particles, have increasing importance as vaccines, delivery vehicles and nanomaterials. However, their use requires stable assemblies. An important cause of loss of stability in proteins is oxidation, which can occur during their production, purification and storage. Despite its importance, very few studies have investigated the effect of oxidation in protein assemblies and their structural units. In this work, we investigated the role of in vitro oxidation in the assembly and stability of rotavirus VP6, a polymorphic protein.

Results: The susceptibility to oxidation of VP6 assembled into nanotubes (VP6NT) and unassembled VP6 (VP6U) was determined and compared to bovine serum albumin (BSA) as control. VP6 was more resistant to oxidation than BSA, as determined by measuring protein degradation and carbonyl content. It was found that assembly protected VP6 from in vitro metal-catalyzed oxidation. Oxidation provoked protein aggregation and VP6NT fragmentation, as evidenced by dynamic light scattering and transmission electron microscopy. Oxidative damage of VP6 correlated with a decrease of its center of fluorescence spectral mass. The in vitro assembly efficiency of VP6U into VP6NT decreased as the oxidant concentration increased.

Conclusions: Oxidation caused carbonylation, quenching, and destruction of aromatic amino acids and aggregation of VP6 in its assembled and unassembled forms. Such modifications affected protein functionality, including its ability to assemble. That assembly protected VP6 from oxidation shows that exposure of susceptible amino acids to the solvent increases their damage, and therefore the protein surface area that is exposed to the solvent is determinant of its susceptibility to oxidation. The inability of oxidized VP6 to assemble into nanotubes highlights the importance of avoiding this modification during the production of proteins that self-assemble. This is the first time that the role of oxidation in protein assembly is studied, evidencing that oxidation should be minimized during the production process if VP6 nanotubes are required.

Show MeSH
Related in: MedlinePlus