SASDD56: Artificially designed de novo protein esPN-Block (FL4) heterocomp...

Basic information

• Entry: Database: SASBDB / ID: SASDD56

Sample

Artificially designed de novo protein esPN-Block (FL4) heterocomplex, e1s2 (FL4)

• stopper PN-Block (WA20) (protein), sPN-Block (WA20), artificially designed de novo protein
• extender PN-Block (FL4) (protein), ePN-Block (FL4), de novo protein

• Biological species: artificially designed de novo protein (others); de novo protein (others)
• Citation: Journal: ACS Synth Biol / Year: 2018; Title: Self-Assembling Supramolecular Nanostructures Constructed from de Novo Extender Protein Nanobuilding Blocks.; Authors: Naoya Kobayashi / Kouichi Inano / Kenji Sasahara / Takaaki Sato / Keisuke Miyazawa / Takeshi Fukuma / Michael H Hecht / Chihong Song / Kazuyoshi Murata / Ryoichi Arai / ; 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.

Contact author

• Ryoichi Arai (Shinshu University)

Models

• Model #1907: ; Type: dummy / Radius of dummy atoms: 2.75 A / Symmetry: P1 / Chi-square value: 0.005 / P-value: 0.235109; Search similar-shape structures of this assembly by Omokage search (details)
• Model #1917: ; Type: atomic; Comment: 2-state model: PDB1: e1s2FL4_Cform.pdb, Rg = 27.65 A, w1 = 0.485; Chi-square value: 0.036143182452; Search similar-shape structures of this assembly by Omokage search (details)
• Model #1918: ; Type: atomic; Comment: 2-state model: PDB2: e1s2FL4_Vform.pdb Rg = 38.66 A, w2 = 0.515; Chi-square value: 0.036143182452; Search similar-shape structures of this assembly by Omokage search (details)
• Model #1912: ; Type: atomic; Comment: MultiFoXS best scoring 5-state model: PDB1, Rg = 39.984, w1 = 0.192; Chi-square value: 0.0125395441575; Search similar-shape structures of this assembly by Omokage search (details)
• Model #1913: ; Type: atomic; Comment: MultiFoXS best scoring 5-state model: PDB2, Rg = 27.7957, w2 = 0.180; Chi-square value: 0.0125395441575; Search similar-shape structures of this assembly by Omokage search (details)
• Model #1914: ; Type: atomic; Comment: MultiFoXS best scoring 5-state model: PDB3, Rg = 28.2499, w3 = 0.305; Chi-square value: 0.0125395441575; Search similar-shape structures of this assembly by Omokage search (details)
• Model #1915: ; Type: atomic; Comment: MultiFoXS best scoring 5-state model: PDB4, Rg = 37.2015, w4 = 0.113; Chi-square value: 0.0125395441575; Search similar-shape structures of this assembly by Omokage search (details)
• Model #1916: ; Type: atomic; Comment: MultiFoXS best scoring 5-state model: PDB5, Rg = 43.0539, w5 = 0.210; Chi-square value: 0.0125395441575; Search similar-shape structures of this assembly by Omokage search (details)

Sample

• Sample: Name: Artificially designed de novo protein esPN-Block (FL4) heterocomplex, e1s2 (FL4); Specimen concentration: 3.5 mg/ml / Entity id: 1029 / 1039
• Buffer: Name: 20 mM HEPES, 100 mM NaCl, 200 mM ArgHCl, 10% glycerol / pH: 7.5

Entity #1029

Name: sPN-Block (WA20) / Type: protein / Description: stopper PN-Block (WA20) / Formula weight: 12.547 / Num. of mol.: 2 / Source: artificially designed de novo protein
Sequence:

MYGKLNKLVE HIKELLQQLN KNWHRHQGNL HDMNQQMEQL FQEFQHFMQG NQDDGKLQNM IHEMQQFMNQ VDNHLQSESD TVHHFHNKLQ ELMNNFHHLV HR

Entity #1039

Name: ePN-Block (FL4) / Type: protein / Description: extender PN-Block (FL4) / Formula weight: 26.879 / Num. of mol.: 1 / Source: de novo protein
Sequence:

MYGKLNKLVE HIKELLQQLN KNWHRHQGNL HDMNQQMEQL FQEFQHFMQG NQDDGKLQNM IHEMQQFMNQ VDNHLQSESD TVHHFHNKLQ ELMNNFHHLV HRKLSGGGGS GGGGSGGGGS GGGGSAAAMY GKLNKLVEHI KELLQQLNKN WHRHQGNLHD MNQQMEQLFQ EFQHFMQGNQ DDGKLQNMIH EMQQFMNQVD NHLQSESDTV HHFHNKLQEL MNNFHHLVHR

Experimental information

• Beam: Instrument name: Photon Factory (PF), High Energy Acceleration Research Organization (KEK) BL-10C; City: Tsukuba / 国: Japan / Type of source: X-ray synchrotronSynchrotron / Wavelength: 0.1488 Å / Dist. spec. to detc.: 1.06 mm
• Detector: Name: Pilatus3 2M / Pixsize x: 0.172 mm

Scan

Title: Artificially designed de novo protein esPN-Block (FL4) heterocomplex, e1s2 (FL4)
Measurement date: Dec 19, 2014 / Cell temperature: 20 °C / Exposure time: 5 sec. / Number of frames: 30 / Unit: 1/nm /

	Min	Max
Q	0.1118	5.4527

Distance distribution function P(R)

Sofotware P(R): GNOM 5.0 / Number of points: 142 /

	Min	Max
Q	0.193045	2.07946
P(R) point	1	142
R	0	12

Result

Type of curve: single_conc /

	Experimental	Standard
MW	40.8 kDa	40.8 kDa

	P(R)	P(R) error	Guinier	Guinier error
Forward scattering, I0	0.02513	0.00121	0.0248	0.0001
Radius of gyration, Rg	3.495 nm	0.243	3.3 nm	0.05

	Min	Max
D	-	12
Guinier point	13	42