SASDD46: Artificially designed de novo protein esPN-Block (HL4) heterocomp...

基本情報

• 登録情報: データベース: SASBDB / ID: SASDD46

試料

Artificially designed de novo protein esPN-Block (HL4) heterocomplex, e1s2 (HL4)

• extender PN-Block (HL4) (protein), ePN-Block (HL4), artificially designed de novo protein
• stopper PN-Block (WA20) (protein), sPN-Block (WA20), artificially designed de novo protein

• 生物種: artificially designed de novo protein (人工物)
• 引用: ジャーナル: ACS Synth Biol / 年: 2018; タイトル: Self-Assembling Supramolecular Nanostructures Constructed from de Novo Extender Protein Nanobuilding Blocks.; 著者: Naoya Kobayashi / Kouichi Inano / Kenji Sasahara / Takaaki Sato / Keisuke Miyazawa / Takeshi Fukuma / Michael H Hecht / Chihong Song / Kazuyoshi Murata / Ryoichi Arai / ; 要旨: 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.

登録者

• Ryoichi Arai (Shinshu University)

モデル

• モデル #1899: ; タイプ: dummy / ダミー原子の半径: 2.75 A / 対称性: P1 / カイ2乗値: 0.004 / P-value: 0.678914; Omokage検索でこの集合体の類似形状データを探す (詳細)
• モデル #1919: ; タイプ: atomic / カイ2乗値: 0.036; Omokage検索でこの集合体の類似形状データを探す (詳細)

試料

• 試料: 名称: Artificially designed de novo protein esPN-Block (HL4) heterocomplex, e1s2 (HL4); 試料濃度: 2.6 mg/ml / Entity id: 1028 / 1029
• バッファ: 名称: 20 mM HEPES, 100 mM NaCl, 200 mM ArgHCl, 10% glycerol; pH: 7.5

要素 #1028

名称: ePN-Block (HL4) / タイプ: protein / 記述: extender PN-Block (HL4) / 分子量: 27.484 / 分子数: 1 / 由来: artificially designed de novo protein
配列:

MYGKLNKLVE HIKELLQQLN KNWHRHQGNL HDMNQQMEQL FQEFQHFMQG NQDDGKLQNM IHEMQQFMNQ VDNHLQSESD TVHHFHNKLQ ELMNNFHHLV HRKLAEAAAK EAAAKEAAAK EAAAKAAAMY GKLNKLVEHI KELLQQLNKN WHRHQGNLHD MNQQMEQLFQ EFQHFMQGNQ DDGKLQNMIH EMQQFMNQVD NHLQSESDTV HHFHNKLQEL MNNFHHLVHR

要素 #1029

名称: sPN-Block (WA20) / タイプ: protein / 記述: stopper PN-Block (WA20) / 分子量: 12.547 / 分子数: 2 / 由来: artificially designed de novo protein
配列:

MYGKLNKLVE HIKELLQQLN KNWHRHQGNL HDMNQQMEQL FQEFQHFMQG NQDDGKLQNM IHEMQQFMNQ VDNHLQSESD TVHHFHNKLQ ELMNNFHHLV HR

実験情報

• ビーム: 設備名称: Photon Factory (PF), High Energy Acceleration Research Organization (KEK) BL-10C; 地域: Tsukuba / 国: Japan / 線源: X-ray synchrotron / 波長: 0.1488 Å / スペクトロメータ・検出器間距離: 1.06 mm
• 検出器: 名称: Pilatus3 2M / Pixsize x: 0.172 mm

スキャン

タイトル: Artificially designed de novo protein esPN-Block (HL4) heterocomplex, e1s2 (HL4)
測定日: 2014年12月19日 / セル温度: 20 °C / 照射時間: 5 sec. / フレーム数: 30 / 単位: 1/nm /

	Min	Max
Q	0.1118	5.3985

距離分布関数 P(R)

ソフトウェア P(R): GNOM 5.0 / ポイント数: 143 /

	Min	Max
Q	0.165951	1.99818
P(R) point	1	143
R	0	15

結果

カーブのタイプ: single_conc /

	実験値	Standard
分子量	58.1 kDa	58.1 kDa

	P(R)	P(R) error	Guinier	Guinier error
前方散乱 I0	0.03619	0.2663	0.035	0.00013
慣性半径, Rg	4.065 nm	0.493	3.62 nm	0.05

	Min	Max
D	-	15
Guinier point	9	36