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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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Dynamic Light scattering (DLS) analysis of 0.4 mg/mL of VP6NT (A) and VP6U (B) exposed to MCO performed with 150 µM of FeCl2 and different H2O2 concentrations for 1 h. Experiments were performed in triplicate, and a representative size distribution for each condition is shown.
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Figure 4: Dynamic Light scattering (DLS) analysis of 0.4 mg/mL of VP6NT (A) and VP6U (B) exposed to MCO performed with 150 µM of FeCl2 and different H2O2 concentrations for 1 h. Experiments were performed in triplicate, and a representative size distribution for each condition is shown.

Mentions: Oxidized samples of VP6NT and VP6U were analyzed by dynamic light scattering (DLS), transmission electron microscopy (TEM), SEC and spectrometry. The size of VP6NT, measured by DLS, decreased as peroxide concentration increased (Figure 4A). The mean hydrodynamic diameter of nanotubes, which was 1,067.0 ± 206.6 nm (corresponding to an equivalent sphere) without oxidation, decreased down to 317.5 ± 40.4 nm after exposition to 10,000 µM H2O2. TEM showed that oxidation provoked the disassembly of nanotubes and aggregation of the resultant unassembled VP6 (Figure 5B). In contrast, oxidation of VP6U resulted in an increase in size, from a hydrodynamic diameter of 7.5 ± 2.6 nm to 2,085.0 ± 289.7 nm after exposition to 10,000 µM of H2O2 (Figure 4B). TEM showed that the increase in size of oxidized VP6U was caused by aggregation (Figure 5C).


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)

Dynamic Light scattering (DLS) analysis of 0.4 mg/mL of VP6NT (A) and VP6U (B) exposed to MCO performed with 150 µM of FeCl2 and different H2O2 concentrations for 1 h. Experiments were performed in triplicate, and a representative size distribution for each condition is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Dynamic Light scattering (DLS) analysis of 0.4 mg/mL of VP6NT (A) and VP6U (B) exposed to MCO performed with 150 µM of FeCl2 and different H2O2 concentrations for 1 h. Experiments were performed in triplicate, and a representative size distribution for each condition is shown.
Mentions: Oxidized samples of VP6NT and VP6U were analyzed by dynamic light scattering (DLS), transmission electron microscopy (TEM), SEC and spectrometry. The size of VP6NT, measured by DLS, decreased as peroxide concentration increased (Figure 4A). The mean hydrodynamic diameter of nanotubes, which was 1,067.0 ± 206.6 nm (corresponding to an equivalent sphere) without oxidation, decreased down to 317.5 ± 40.4 nm after exposition to 10,000 µM H2O2. TEM showed that oxidation provoked the disassembly of nanotubes and aggregation of the resultant unassembled VP6 (Figure 5B). In contrast, oxidation of VP6U resulted in an increase in size, from a hydrodynamic diameter of 7.5 ± 2.6 nm to 2,085.0 ± 289.7 nm after exposition to 10,000 µM of H2O2 (Figure 4B). TEM showed that the increase in size of oxidized VP6U was caused by aggregation (Figure 5C).

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