Effect of Chemical Oxidation on the Higher Order Structure, Stability, Aggregation, and Biological Function of Interferon Alpha-2a: Role of Local Structural Changes Detected by 2D NMR
Effect of Chemical Oxidation on the Higher Order Structure, Stability, Aggregation, and Biological Function of Interferon Alpha-2a: Role of Local Structural Changes Detected by 2D NMR
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DOI：
10.1007/s11095-018-2518-y
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发表时间：
2018-12-01
影响因子：
3.7
通讯作者：
Mallela, Krishna M. G.
中科院分区：
文献类型：
--
作者：
Shah, Dinen D.;Singh, Surinder M.;Mallela, Krishna M. G.
目的氧化型干扰素具有聚集性和免疫原性。本研究探讨了氧化诱导干扰素α-2a（IFN-α 2a）聚集和功能丧失的结构机制。氧化的蛋白质探测使用生物物理方法，包括变性剂熔体，颗粒计数，蛋白水解偶联质谱，和2D NMR.ResultsOxidized IFNA 2a没有表现出重大变化，其二级结构，但在三级结构中表现出微小的变化时相比，未氧化的蛋白质。此外，在氧化后观察到构象稳定性的显著损失。相应地，观察到蛋白质聚集增加，导致形成不溶性微粒。氧化蛋白在其抗病毒效力和细胞病变抑制效力方面显示出降低的生物学功能。蛋白水解偶联质谱鉴定了5个被氧化的蛋氨酸残基，氧化程度与其可及表面积之间无相关性。2D N-15-H-1 HSQC NMR确定残留水平的局部结构变化的蛋白质氧化后，这是无法检测到的全球探针，如远紫外圆二色性和fluorescence.ConclusionsIncreased蛋白质聚集和降低功能的IFNA 2a氧化后与该网站的修改确定的蛋白水解耦合质谱和局部结构变化的蛋白质检测2D NMR。
PurposeOxidized interferons have been shown to aggregate and cause immunogenicity. In this study, the structural mechanisms underlying oxidation-induced interferon alpha-2a (IFNA2a) aggregation and loss of function were examined.MethodsIFNA2a was oxidized using 0.037% vol/vol hydrogen peroxide. Oxidized protein was probed using biophysical methods that include denaturant melts, particle counting, proteolysis-coupled mass spectrometry, and 2D NMR.ResultsOxidized IFNA2a did not show major changes in its secondary structure, but showed minor changes in tertiary structure when compared to the unoxidized protein. In addition, a significant loss of conformational stability was observed upon oxidation. Correspondingly, increased protein aggregation was observed resulting in the formation of sub-visible particles. Oxidized protein showed decreased biological function in terms of its anti-viral potency and cytopathic inhibition efficacy. Proteolysis-coupled mass spectrometry identified five methionine residues that were oxidized with no correlation between the extent of oxidation and their accessible surface area. 2D N-15-H-1 HSQC NMR identified residue-level local structural changes in the protein upon oxidation, which were not detectable by global probes such as far-UV circular dichroism and fluorescence.ConclusionsIncreased protein aggregation and decreased function of IFNA2a upon oxidation correlated with the site of modification identified by proteolysis-coupled mass spectrometry and local structural changes in the protein detected by 2D NMR.