TY - JOUR
T1 - Self-Assembling Supramolecular Nanostructures Constructed from de Novo Extender Protein Nanobuilding Blocks
AU - Kobayashi, Naoya
AU - Inano, Kouichi
AU - Sasahara, Kenji
AU - Sato, Takaaki
AU - Miyazawa, Keisuke
AU - Fukuma, Takeshi
AU - Hecht, Michael H.
AU - Song, Chihong
AU - Murata, Kazuyoshi
AU - Arai, Ryoichi
N1 - Funding Information:
We thank Prof. Nobuyasu Koga, Dr. Rie Koga, and Dr. Takahiro Kosugi at the Institute for Molecular Science (IMS) for assistance in SEC-MALS experiments. We are grateful to Prof. Teruyuki Nagamune at University of Tokyo for kindly providing the plasmids pET32/EBFP-linker-EGFP. We also thank Prof. Nobuaki Hayashida at Shinshu University and Dr. Shinya Honda at Advanced Industrial Science and Technology for helpful advice. This work was supported by Joint Research of IMS and the Bio-AFM summer school at Kanazawa University. We thank Dr. Nobutaka Shimizu, Dr. Noriyuki Igarashi and Photon Factory (PF) staff for their help in synchrotron SAXS experiments which were performed at PF, KEK, under the approval of PF program advisory committee (Proposal Nos. 2014G111, 2016G153, and 2016G606). We are indebted to Divisions of Gene Research and Instrumental Analysis of Research Center for Supports to Advanced Science, Shinshu University for providing facilities. This work was supported by JSPS Research Fellowships (DC2) and JSPS KAKENHI Grant Nos. JP14J10185 and JP16H06837 to N.K., and JSPS KAKENHI Grant Nos. JP22113508, JP24113707 (Innovative Areas Intrinsically Disordered Proteins), JP24780097 JP26288101, JP16K05841, JP16H00761 (Innovative Areas Dynamical Ordering & Integrated Functions), JSPS Postdoctoral Fellowships for Research Abroad, and Program for Dissemination of Tenure-Track System, to R.A. The work was also supported by NSF Grants MCB-1050510 and MCB-1409402 to M.H.H.
Funding Information:
We thank Prof. Nobuyasu Koga, Dr. Rie Koga, and Dr. Takahiro Kosugi at the Institute for Molecular Science (IMS) for assistance in SEC−MALS experiments. We are grateful to Prof. Teruyuki Nagamune at University of Tokyo for kindly providing the plasmids pET32/EBFP-linker-EGFP. We also thank Prof. Nobuaki Hayashida at Shinshu University and Dr. Shinya Honda at Advanced Industrial Science and Technology for helpful advice. This work was supported by Joint Research of IMS and the Bio-AFM summer school at Kanazawa University. We thank Dr. Nobutaka Shimizu, Dr. Noriyuki Igarashi, and Photon Factory (PF) staff for their help in synchrotron SAXS experiments, which were performed at PF, KEK, under the approval of PF program advisory committee (Proposal Nos. 2014G111, 2016G153, and 2016G606). We are indebted to Divisions of Gene Research and Instrumental Analysis of Research Center for Supports to Advanced Science, Shinshu University, for providing facilities. This work was supported by JSPS Research Fellowships (DC2) and JSPS KAKENHI Grant Nos. JP14J10185 and JP16H06837 to N.K., and JSPS KAKENHI Grant Nos. JP22113508, JP24113707 (Innovative Areas “Intrinsically Disordered Proteins”), JP24780097, JP26288101, JP16K05841, JP16H00761 (Innovative Areas “Dynamical Ordering & Integrated Functions”), JSPS Postdoctoral Fellowships for Research Abroad, and Program for Dissemination of Tenure-Track System, to R.A. The work was also supported by NSF Grants MCB-1050510 and MCB-1409402 to M.H.H.
Publisher Copyright:
Copyright © 2018 American Chemical Society.
PY - 2018/5/18
Y1 - 2018/5/18
N2 - 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.
AB - 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.
KW - de novo protein
KW - nanostructure
KW - protein engineering
KW - protein nanobuilding block
KW - protein-based supramolecular polymers
KW - self-assembly
UR - http://www.scopus.com/inward/record.url?scp=85046466746&partnerID=8YFLogxK
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U2 - 10.1021/acssynbio.8b00007
DO - 10.1021/acssynbio.8b00007
M3 - Article
C2 - 29690759
AN - SCOPUS:85046466746
SN - 2161-5063
VL - 7
SP - 1381
EP - 1394
JO - ACS Synthetic Biology
JF - ACS Synthetic Biology
IS - 5
ER -