A feasibility study of inverse contrast-matching small-angle neutron scattering method combined with size exclusion chromatography using antibody interactions as model systems
A feasibility study of inverse contrast-matching small-angle neutron scattering method combined with size exclusion chromatography using antibody interactions as model systems
复制标题
以抗体相互作用为模型系统的逆对比匹配小角中子散射法与尺寸排阻色谱相结合的可行性研究
DOI：
10.1093/jb/mvab012
复制
发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Kato Koichi
中科院分区：
文献类型：
--
作者：
Sato Nobuhiro;Yogo Rina;Yanaka Saeko;Martel Anne;Porcar Lionel;Morishima Ken;Inoue Rintaro;Tominaga Taiki;Arimori Takao;Takagi Junichi;Sugiyama Masaaki;Kato Koichi
小角中子散射（SANS）和小角X射线散射（SAXS）是表征生物分子复合物结构的有力技术。特别是，SANS能够通过对比度匹配技术的选择性氘化来选择性地观察复合物中的特定组分。然而，在大多数情况下，具有异质寡聚体的生物分子相互作用系统通常包含不利的聚集体和未结合的物种，妨碍数据解释。为了克服这些问题，SAXS最近已与尺寸排阻色谱法（SEC）相结合，这使得能够在多组分系统中分离目标复合物。相比之下，SEC-SANS仅处于初步阶段。因此，我们在此进行了可行性研究，这种方法的基础上，我们新开发的反向对比度匹配（iCM）SANS技术，使用抗体相互作用作为模型系统。将免疫球蛋白G（IgG）或其Fc片段与75%氘代Fc结合蛋白（即化脓性链球菌IgG降解酶的突变形式和Fcγ受体IIIb的可溶形式）混合，并进行SEC-SANS和SEC-SAXS作为参考。我们成功地观察到来自与这些结合配偶体复合形成的非氘代IgG或Fc的SANS，这在D2 O中的SANS方面是不可观察的，因此证明了SEC-iCM-SANS方法的潜在实用性。
Small-angle neutron scattering (SANS) and small- angle X-ray scattering (SAXS) are powerful techniques for the structural characterization of biomolecular complexes. In particular, SANS enables a selective observation of specific components in complexes by selective deuteration with contrast-matching techniques. In most cases, however, biomolecular interaction systems with heterogeneous oligomers often contain unfavorable aggregates and unbound species, hampering data interpretation. To overcome these problems, SAXS has been recently combined with size exclusion chromatography (SEC), which enables the isolation of the target complex in a multi-component system. By contrast, SEC–SANS is only at a preliminary stage. Hence, we herein perform a feasibility study of this method based on our newly developed inverse contrast-matching (iCM) SANS technique using antibody interactions as model systems. Immunoglobulin G (IgG) or its Fc fragment was mixed with 75% deuterated Fc-binding proteins,i.e.a mutated form of IgG-degrading enzyme ofStreptococcus pyogenesand a soluble form of Fcγ receptor IIIb, and subjected to SEC–SANS as well as SEC–SAXS as reference. We successfully observe SANS from the non-deuterated IgG or Fc formed in complex with these binding partners, which were unobservable in terms of SANS in D2O, hence demonstrating the potential utility of the SEC–iCM–SANS approach.