Self-Assembling Supramolecular Nanostructures Constructed from de Novo Extender Protein Nanobuilding Blocks
Self-Assembling Supramolecular Nanostructures Constructed from de Novo Extender Protein Nanobuilding Blocks
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DOI：
10.1021/acssynbio.8b00007
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发表时间：
2018-05-01
影响因子：
4.7
通讯作者：
Arai, Ryoichi
中科院分区：
文献类型：
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作者：
Kobayashi, Naoya;Inano, Kouichi;Arai, Ryoichi
设计新型蛋白质自组装成超分子复合物对于纳米生物技术和合成生物学的发展非常重要。最近，我们通过融合分子间折叠的二聚体从头WA 20蛋白和T4噬菌体纤维蛋白的三聚体折叠子结构域，设计并创建了蛋白质纳米结构单元（PN-单元），WA 20-折叠子（小林等人，J. Am. 2015，137，11285）。WA 20-折叠子形成了几种类型的自组装纳米结构的6聚体的倍数，包括桶状六聚体和四面体状十二聚体。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二进制模式的WA 20蛋白质与各种接头来设计从头扩展蛋白纳米构建块（ePN-块）。在大肠杆菌可溶性组分中表达具有长螺旋接头或柔性接头的ePN-Blocks，并通过非变性PAGE、尺寸排阻色谱-多角度光散射（SEC-MALS）、小角X射线散射（SAXS）和透射电子显微镜分析纯化的ePN-Blocks。这些结果表明形成了各种结构的同源寡聚物。随后，我们重建异源寡聚复合物的延伸和终止PN块变性和重折叠。目前的SEC MALS和SAXS分析表明，延长剂和终止剂PN-嵌段（esPN-嵌段）杂合物形成不同类型的延长链样构象，这取决于它们的接头类型。此外，在液体中的原子力显微镜成像表明，esPN-Block杂配合物与金属离子进一步自组装成云母表面的超分子纳米结构。总之，本数据表明，使用从头蛋白质的自组装PN-块的设计和构建是用于构建超分子蛋白质复合物的聚合物纳米结构的有用策略。
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.