The design of novel proteins that self-assemble into supramolecular complexes is important for development in nanobiotechnology and synthetic biology. Recently, we designed and created a protein nanobuilding block (PN–Block), WA20-foldon, by fusing an intermolecularly folded dimeric de novo WA20 protein and a trimeric foldon domain of T4 phage fibritin (Kobayashi et al., J. Am. Chem. Soc.2015, 137, 11285). WA20-foldon formed several types of self-assembling nanoarchitectures in multiples of 6-mers, including a barrel-like hexamer and a tetrahedron-like dodecamer. In this study, to construct chain-like polymeric nanostructures, we designed de novo extender protein nanobuilding blocks (ePN–Blocks) by tandemly fusing two de novo binary-patterned WA20 proteins with various linkers. The ePN–Blocks with long helical linkers or flexible linkers were expressed in soluble fractions of Escherichia coli, and the purified ePN–Blocks were analyzed by native PAGE, size exclusion chromatography–multiangle light scattering (SEC–MALS), small-angle X-ray scattering (SAXS), and transmission electron microscopy. These results suggest formation of various structural homo-oligomers. Subsequently, we reconstructed hetero-oligomeric complexes from extender and stopper PN–Blocks by denaturation and refolding. The present SEC–MALS and SAXS analyses show that extender and stopper PN–Block (esPN–Block) heterocomplexes formed different types of extended chain-like conformations depending on their linker types. Moreover, atomic force microscopy imaging in liquid suggests that the esPN–Block heterocomplexes with metal ions further self-assembled into supramolecular nanostructures on mica surfaces. Taken together, the present data demonstrate that the design and construction of self-assembling PN–Blocks using de novo proteins is a useful strategy for building polymeric nanoarchitectures of supramolecular protein complexes.

中文翻译：

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从de Novo延伸蛋白纳米构造块构造的自组装超分子纳米结构。

自组装成超分子复合物的新型蛋白质的设计对于纳米生物技术和合成生物学的发展很重要。最近，我们通过融合分子间折叠的二聚从头WA20蛋白和T4噬菌体纤维蛋白的三聚体foldon结构域，设计并创建了一个蛋白质纳米构件（PN-Block）WA20- foldon（Kobayashi et al。，J.会志2015年，137，11285）。WA20-foldon以六聚体的倍数形成了几种类型的自组装纳米结构，包括桶状六聚体和四面体十二聚体。在这项研究中，为了构建链状聚合物纳米结构，我们通过串联融合两个从头开始的增量剂蛋白质纳米结构单元（ePN–Blocks）从头开始具有各种接头的二元模式化WA20蛋白。具有长螺旋接头或柔性接头的ePN-Blocks在大肠杆菌的可溶性级分中表达，并通过天然PAGE，尺寸排阻色谱-多角度光散射（SEC-MALS），小角度X射线散射（SAXS）和透射电子显微镜对纯化的ePN-Blocks进行了分析。这些结果表明形成了各种结构同型低聚物。随后，我们通过变性和重折叠从增量剂和终止子PN-Blocks重建了异寡聚复合物。目前的SEC-MALS和SAXS分析表明，取决于连接子的类型，增量剂和终止剂PN-Block（esPN-Block）杂合物形成了不同类型的扩展链状构象。此外，液体中的原子力显微镜成像表明，带有金属离子的esPN-Block杂合物进一步自组装成云母表面的超分子纳米结构。在一起从头蛋白质是构建超分子蛋白质复合物的聚合物纳米结构的有用策略。