Abstract
BackgroundProtein 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.ResultsThe 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.ConclusionsOxidation 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.
Highlights
Protein assemblies, such as virus-like particles, have increasing importance as vaccines, delivery vehicles and nanomaterials
Size exclusion chromatography (SEC) analysis showed two populations (Figure 1B), one that migrated at the column exclusion limit, which corresponded to VP6 nanotubes (VP6NT), and a second one with smaller size identified as Unassembled VP6 (VP6U), containing unassembled VP6 monomers and trimers
Intrinsic fluorescence spectra of aromatic amino acids were acquired for VP6 assembled into nanotubes (VP6NT) and VP6U at 280 (Trp and Tyr, Figure 1D) and 295 nm (Trp)
Summary
Protein assemblies, such as virus-like particles, have increasing importance as vaccines, delivery vehicles and nanomaterials. Viral proteins have a primary role in the field, as many of them are capable of self-assemble to form macromolecular structures with unique properties, such as virus-like particles (VLP) and other assemblies. Oxidation provoked VLP aggregation and cross-linking of S protein chains, leading to a complete loss of antigenicity Such studies highlight the importance of further investigating the effect of aggregation on protein assemblies
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