Self-Assembling Supramolecular Nanostructures Constructed from de Novo Extender Protein Nanobuilding Blocks

Naoya Kobayashi, Kouichi Inano, Kenji Sasahara, Takaaki Sato, Keisuke Miyazawa, Takeshi Fukuma, Michael H. Hecht, Chihong Song, Kazuyoshi Murata, Ryoichi Arai

Research output: Contribution to journalArticle

9 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)1381-1394
Number of pages14
JournalACS Synthetic Biology
Volume7
Issue number5
DOIs
StatePublished - May 18 2018

All Science Journal Classification (ASJC) codes

  • Biomedical Engineering
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)

Keywords

  • de novo protein
  • nanostructure
  • protein engineering
  • protein nanobuilding block
  • protein-based supramolecular polymers
  • self-assembly

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    Kobayashi, N., Inano, K., Sasahara, K., Sato, T., Miyazawa, K., Fukuma, T., Hecht, M. H., Song, C., Murata, K., & Arai, R. (2018). Self-Assembling Supramolecular Nanostructures Constructed from de Novo Extender Protein Nanobuilding Blocks. ACS Synthetic Biology, 7(5), 1381-1394. https://doi.org/10.1021/acssynbio.8b00007