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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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Characterization of purified VP6NT. A) 12% SDS-PAGE gel stained with Coomassie Blue (under reducing conditions): Lane 1, Molecular weight marker Benchmark (Life Technologies Corp., Carlsbad, CA, USA), the more intense band corresponds to 50 KDa. Lane 2, VP6NT. B) Size exclusion chromatography, Peak 1 corresponds to VP6NT, peak 2 to VP6U and peak 3 to salts. KD refers to the relative elution volume calculated with Equation 1. C) Transmission electronic micrograph at 85,000X. D) Intrinsic fluorescence spectrum of 40 µg/mL of VP6NT or VP6U. Excitation at 280 nm.
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Figure 1: Characterization of purified VP6NT. A) 12% SDS-PAGE gel stained with Coomassie Blue (under reducing conditions): Lane 1, Molecular weight marker Benchmark (Life Technologies Corp., Carlsbad, CA, USA), the more intense band corresponds to 50 KDa. Lane 2, VP6NT. B) Size exclusion chromatography, Peak 1 corresponds to VP6NT, peak 2 to VP6U and peak 3 to salts. KD refers to the relative elution volume calculated with Equation 1. C) Transmission electronic micrograph at 85,000X. D) Intrinsic fluorescence spectrum of 40 µg/mL of VP6NT or VP6U. Excitation at 280 nm.

Mentions: A typical VP6 nanotube preparation was characterized. The purity of VP6 was confirmed in reducing denaturing SDS-PAGE gels, which showed a single band with the molecular weight reported for VP6 (Figure 1A). Size exclusion chromatography (SEC) analysis showed two populations (Figure 1B), one that migrated at the column exclusion limit (peak 1), which corresponded to VP6 nanotubes (VP6NT), and a second one with smaller size identified as VP6U (peak 2), containing unassembled VP6 monomers and trimers. The population containing VP6NT constituted 95% of the total protein, which is a typical value obtained with this purification process [3,29]. The presence of VP6NT structures was confirmed by TEM (Figure 1C). Intrinsic fluorescence spectra of aromatic amino acids were acquired for VP6NT and VP6U at 280 (Trp and Tyr, Figure 1D) and 295 nm (Trp). VP6U had a fluorescence quantum yield 2 and 2.5 times higher than VP6NT, at 280 and 295 nm, respectively.


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)

Characterization of purified VP6NT. A) 12% SDS-PAGE gel stained with Coomassie Blue (under reducing conditions): Lane 1, Molecular weight marker Benchmark (Life Technologies Corp., Carlsbad, CA, USA), the more intense band corresponds to 50 KDa. Lane 2, VP6NT. B) Size exclusion chromatography, Peak 1 corresponds to VP6NT, peak 2 to VP6U and peak 3 to salts. KD refers to the relative elution volume calculated with Equation 1. C) Transmission electronic micrograph at 85,000X. D) Intrinsic fluorescence spectrum of 40 µg/mL of VP6NT or VP6U. Excitation at 280 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Characterization of purified VP6NT. A) 12% SDS-PAGE gel stained with Coomassie Blue (under reducing conditions): Lane 1, Molecular weight marker Benchmark (Life Technologies Corp., Carlsbad, CA, USA), the more intense band corresponds to 50 KDa. Lane 2, VP6NT. B) Size exclusion chromatography, Peak 1 corresponds to VP6NT, peak 2 to VP6U and peak 3 to salts. KD refers to the relative elution volume calculated with Equation 1. C) Transmission electronic micrograph at 85,000X. D) Intrinsic fluorescence spectrum of 40 µg/mL of VP6NT or VP6U. Excitation at 280 nm.
Mentions: A typical VP6 nanotube preparation was characterized. The purity of VP6 was confirmed in reducing denaturing SDS-PAGE gels, which showed a single band with the molecular weight reported for VP6 (Figure 1A). Size exclusion chromatography (SEC) analysis showed two populations (Figure 1B), one that migrated at the column exclusion limit (peak 1), which corresponded to VP6 nanotubes (VP6NT), and a second one with smaller size identified as VP6U (peak 2), containing unassembled VP6 monomers and trimers. The population containing VP6NT constituted 95% of the total protein, which is a typical value obtained with this purification process [3,29]. The presence of VP6NT structures was confirmed by TEM (Figure 1C). Intrinsic fluorescence spectra of aromatic amino acids were acquired for VP6NT and VP6U at 280 (Trp and Tyr, Figure 1D) and 295 nm (Trp). VP6U had a fluorescence quantum yield 2 and 2.5 times higher than VP6NT, at 280 and 295 nm, respectively.

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