Castro-Acosta Ricardo M, Rodríguez-Limas William A, Valderrama Brenda, Ramírez Octavio T, Palomares Laura A
Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, A,P, 510-3, C,P, 62210, Cuernavaca, Morelos, Mexico.
Microb Cell Fact. 2014 Feb 17;13(1):25. doi: 10.1186/1475-2859-13-25.
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.
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.
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.
蛋白质组装体，如病毒样颗粒，作为疫苗、递送载体和纳米材料的重要性日益增加。然而，它们的使用需要稳定的组装体。蛋白质稳定性丧失的一个重要原因是氧化，这可能发生在其生产、纯化和储存过程中。尽管其重要性，但很少有研究调查氧化对蛋白质组装体及其结构单元的影响。在这项工作中，我们研究了体外氧化在轮状病毒VP6（一种多态性蛋白质）的组装和稳定性中的作用。
测定了组装成纳米管的VP6（VP6NT）和未组装的VP6（VP6U）对氧化的敏感性，并与作为对照的牛血清白蛋白（BSA）进行比较。通过测量蛋白质降解和羰基含量确定，VP6比BSA更耐氧化。发现组装可保护VP6免受体外金属催化的氧化。动态光散射和透射电子显微镜证明，氧化引发蛋白质聚集和VP6NT碎片化。VP6的氧化损伤与其荧光光谱质量中心的降低相关。随着氧化剂浓度增加，VP6U体外组装成VP6NT的效率降低。
氧化导致VP6组装和未组装形式的羰基化、淬灭、芳香族氨基酸破坏以及聚集。这些修饰影响蛋白质功能，包括其组装能力。组装保护VP6免受氧化表明，易感氨基酸暴露于溶剂会增加其损伤，因此暴露于溶剂的蛋白质表面积决定了其氧化敏感性。氧化的VP6无法组装成纳米管突出了在自组装蛋白质生产过程中避免这种修饰的重要性。这是首次研究氧化在蛋白质组装中的作用，证明如果需要VP6纳米管，在生产过程中应尽量减少氧化。
