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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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TEM micrographs of VP6U subjected to assembly conditions after exposure for 1 h to MCO. A) Nanotubes obtained after assembly of untreated VP6U. Other panels, MCO with B) 100 µM C) 250 µM. D) 500 µM. E) 1,000 µM. F) 5,000 µM of H2O2. Samples were stained with 2% uranyl acetate and observed at a magnification of 85,000X.
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Figure 9: TEM micrographs of VP6U subjected to assembly conditions after exposure for 1 h to MCO. A) Nanotubes obtained after assembly of untreated VP6U. Other panels, MCO with B) 100 µM C) 250 µM. D) 500 µM. E) 1,000 µM. F) 5,000 µM of H2O2. Samples were stained with 2% uranyl acetate and observed at a magnification of 85,000X.

Mentions: Oxidized VP6U at various H2O2 concentrations was subjected to in vitro assembly conditions to determine if oxidation impedes its assembly into VP6NT. Samples incubated for 6 h were analyzed by SEC to determine the relative concentration of assembled VP6, which elutes with the void volume of the SEC column (KD = 0) (Figure 8A). A sample of VP6U not treated for assembly is shown for comparison. VP6U eluted at a KD of 0.6. The peak corresponding to VP6U disappeared in all samples treated for assembly, and peaks appeared at lower KD. The abundance of the peak corresponding to VP6NT decreased as H2O2 concentration increased. Assembly efficiencies were determined by SEC and calculated by dividing the area of the peak at 0 KD (corresponding to VP6NT) by the total peak area below 0.8 KD, to exclude the salt peak (Figure 8A). VP6U before being subjected to the assembly reactions is shown for comparison. Untreated VP6 assembled into VP6NT with a 72% efficiency. Nanotubes with the expected characteristic were observed by TEM (Figure 9A). The assembly efficiency of oxidized VP6U decreased as H2O2 concentration increased (Figure 8B), and the quality of the assembled VP6NT decreased (Figure 9B to F). It was increasingly difficult to find assembled nanotubes as the H2O2 concentration increased, and those found had various defects, such as fractures, incomplete NT and association with aggregates (Figure 9B to F). Most likely the assembly efficiency was overestimated, as aggregation of VP6U upon oxidation was observed.


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)

TEM micrographs of VP6U subjected to assembly conditions after exposure for 1 h to MCO. A) Nanotubes obtained after assembly of untreated VP6U. Other panels, MCO with B) 100 µM C) 250 µM. D) 500 µM. E) 1,000 µM. F) 5,000 µM of H2O2. Samples were stained with 2% uranyl acetate and observed at a magnification of 85,000X.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 9: TEM micrographs of VP6U subjected to assembly conditions after exposure for 1 h to MCO. A) Nanotubes obtained after assembly of untreated VP6U. Other panels, MCO with B) 100 µM C) 250 µM. D) 500 µM. E) 1,000 µM. F) 5,000 µM of H2O2. Samples were stained with 2% uranyl acetate and observed at a magnification of 85,000X.
Mentions: Oxidized VP6U at various H2O2 concentrations was subjected to in vitro assembly conditions to determine if oxidation impedes its assembly into VP6NT. Samples incubated for 6 h were analyzed by SEC to determine the relative concentration of assembled VP6, which elutes with the void volume of the SEC column (KD = 0) (Figure 8A). A sample of VP6U not treated for assembly is shown for comparison. VP6U eluted at a KD of 0.6. The peak corresponding to VP6U disappeared in all samples treated for assembly, and peaks appeared at lower KD. The abundance of the peak corresponding to VP6NT decreased as H2O2 concentration increased. Assembly efficiencies were determined by SEC and calculated by dividing the area of the peak at 0 KD (corresponding to VP6NT) by the total peak area below 0.8 KD, to exclude the salt peak (Figure 8A). VP6U before being subjected to the assembly reactions is shown for comparison. Untreated VP6 assembled into VP6NT with a 72% efficiency. Nanotubes with the expected characteristic were observed by TEM (Figure 9A). The assembly efficiency of oxidized VP6U decreased as H2O2 concentration increased (Figure 8B), and the quality of the assembled VP6NT decreased (Figure 9B to F). It was increasingly difficult to find assembled nanotubes as the H2O2 concentration increased, and those found had various defects, such as fractures, incomplete NT and association with aggregates (Figure 9B to F). Most likely the assembly efficiency was overestimated, as aggregation of VP6U upon oxidation was observed.

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