Photon Factory Activity Report 2016 #34 (2017)

BL-10C, BL-6A/2014G111, 2016G153, 2016G617

Small-Angle X-ray Scattering Analyses of Self-Assembling Chain-like Nanostructures
Constructed from de Novo Extender Protein Nanobuilding Blocks
Naoya Kobayashi,1 Kouichi Inano,1 Kenji Sasahara,1 Takaaki Sato,1 and Ryoichi Arai1,2,*
1Fac. of Tex. Sci. Tech., Shinshu Univ., Ueda, Nagano 386-8567, Japan
2Res. Ctr. for Fungal & Microb. Dynam., Shinshu Univ., Minamiminowa, Nagano 399-4598, Japan
1 Introduction
The design of novel proteins that self-assemble intosupramolecular complexes is important for development
in synthetic biology and nanobiotechnology [1]. Several
years ago, we reported an intermolecularly folded dimeric
4-helix bundle structure of WA20 [2], a de novo protein
created by the binary code strategy [3,4]. To harness theunusual intertwined structure of WA20 for the selfassembly of supramolecular nanostructures, we recently
designed and created a protein nanobuilding block (PNBlock), WA20-foldon, by fusing the intermolecularly
folded dimeric de novo WA20 protein and the trimericfoldon domain of T4 phage fibritin [5]. The WA20-foldon
formed several distinctive types of self-assemblingnanoarchitectures in multiples of 6-mers, including abarrel-like-shaped hexamer and a tetrahedron-like-shaped
dodecamer.
In the present study, we designed and created de novo
extender protein nanobuilding blocks (ePN-Blocks) bytandemly fusing two de novo WA20 proteins withdifferent linkers [6,7], and generated a new series of PNBlocks to construct self-assembling chain-likenanostructures (Fig. 1).
A
Homooligomers

Self-assembly

FusionWA20＋Helical linker or Flexible linker

WA20-Linker-WA20
Extender Protein Nanobuilding Blocks(ePN-Blocks)

B ePN-Blocks with helical linker (HL)

CePN-Blocks with flexible linker (FL)

＝ EAAAKCCN

HL4＝ GGGGS
FL4

N

Fig. 1: Schematics of extender protein nanobuilding
blocks (ePN-Blocks). (A) Construction and assembly ofePN-Blocks; ribbon representation and schematics of theintermolecularly folded dimeric de novo protein WA20
(PDB code 3VJF) [2] are shown in red and blue. The
ePN-Blocks were constructed by tandemly fusing two denovo WA20 proteins using various linkers [6,7]. Helicaland flexible linkers are shown as a yellow rods and a
black lines, respectively. (B) Schematics of ePN-Blocks
with helical linkers (HL4). (C) Schematics of ePN-Blocks
with flexible linkers (FL4).
2 Experiment
The ePN-Block proteins were expressed in E. coli
BL21 Star (DE3) (Invitrogen) harboring pET-WA20-

Linker-WA20 in LB broth containing 50 µg/mL
ampicillin at 37°C. Protein expression was induced using
0.2 mM IPTG. Proteins were extracted from harvested
cells by sonication in lysis buffer. Proteins were thenpurified using immobilized metal ion affinity
chromatography (IMAC) with TALON metal affinity
resin (Clontech, Takara Bio). After IMAC purification,
concentrated ePN-Block samples were fractionated in 20
mM HEPES buffer (pH 7.5) containing 100 mM NaCl,
10% glycerol, and 200 mM ArgHCl using size exclusion
chromatography (SEC) on a HiLoad 16/600 Superdex
200 pg (GE healthcare) column.
SAXS measurements were performed on several
fractions of ePN-Block (HL4 or FL4) homooligomers
after separation by SEC and chicken egg white lysozyme(Wako Pure Chemical Industries) in 20 mM HEPES
buffer (pH 7.5) containing 100 mM NaCl, 200 mM
ArgHCl, and 10% glycerol at 20°C using synchrotron
radiation (λ, 0.1488 nm) with a PILATUS3 2M detector(Dectris) at the KEK Photon Factory BL-10C beamline.
Two-dimensional scattering images were integrated into
one-dimensional scattering intensities I(q) as a function of
the magnitude of the scattering vector q = (4π/λ) sin(θ/2)
using the FIT2D program, where θ is the total scattering
angle. In the present experiments, the structure factor wasalmost at unity (I(q) ≈ n P(q)), because interparticle
interactions such as the excluded volume effect andelectrostatic interactions can be neglected at low protein
and high salt concentrations. The indirect Fouriertransformation (IFT) technique was used to calculate pairdistance distribution functions, p(r) for particles with avirtually model-free routine. Forward scattering intensity
I(q→0) was extrapolated from SAXS data, and the radiusof gyration Rg was estimated using the Guinier
approximation.
3 Results and Discussion
We performed experiments using soluble samples ofePN-Blocks with HL4 and FL4. After purification ofIMAC followed by SEC, the fractionated samples ofePN-Block (HL4) and ePN-Block (FL4) homooligomers
were analyzed by SAXS (Fig. 2 and Table 1) and SECMALS (Table 2). In Figures 2A and 2B, SAXS intensitiesof ePN-Block homooligomers samples are shown withchicken egg lysozyme as a molecular mass reference
standard (Mw, 14.3 kDa). Assuming that these proteins
have practically identical scattering length densities andspecific volumes, and the structure factor S(q) ≈ 1 forscreened electrostatic repulsion in dilute samples, thePhoton Factory Activity Report 2016 #34 (2017)

forward scattering intensity normalized by protein
concentration I(q → 0)/c is proportional to the weightaverage molecular mass (Mw). The Mw values of thesesamples (Table 1) are roughly consistent with SECMALS analyses (Table 2). These data indicate thepresence of homooligomeric species constructed fromePN-Blocks, such as dimer, trimer, tetramer, andpentamer, because the molecular mass of a protmer is ~27
kDa. Pair-distance distribution functions p(r) were
determined using IFT (Figs. 2C and 2D). The integral ofp(r)/c from r = 0 to r = Dmax is equal to the extrapolated
forward absolute scattering intensity normalized byconcentration (I(q→0)/c) and is therefore proportional toMw. Accordingly, the sizes of both p(r) series of ePNBlock (HL4) and ePN-Block (FL4) homooligomer
samples (Figs. 2C and 2D) increase with the Mw values ofthese samples, suggesting that the sizes of chain-likenanostructures increase with multimeric numbers of ePNBlocks. In addition, we have tried to do SEC-SAXS
experiments, and further analyses are in progress.
Acknowledgement
We thank the PF staff for assistance in X-ray scatteringexperiments and Dr. N. Koga and Dr. R. Koga at Institutefor Molecular Science (IMS) for assistance in SECMALS. This work was supported by MEXT/JSPS
KAKENHI Grant Numbers JP24113707, JP24780097,
JP14J10185, JP16K05841 JP16H06837, JP16H00761 andJSPS Research Fellowships. This work was also
supported by the Joint Studies Program of IMS.

ACB

DFig. 2: SAXS analyses of the ePN-Block homooligomersamples. Concentration-normalized absolute scattering
intensities I(q)/c of (A) ePN-Block (HL4), e(HL4),
homooligomer samples and (B) ePN-Block (FL4), e(FL4),
homooligomer samples. Chicken egg lysozyme was used
as a molecular mass reference standard. Their real-space
information, concentration-normalized pair-distance
distribution functions p(r)/c of (C) ePN-Block (HL4)

homooligomer samples and (D) ePN-Block (FL4)homooligomer samples, which were calculated using IFT.
Table 1: SAXS analyses of ePN-Block homooligomersePN-Block
I(q→0)/cMw
RgDmaxhomooligomers [cm-1mg-1mL] [nm][nm] [kDa]e(HL4), (A)0.05936.1

2580.9e(HL4), (B)0.05335.7

2372.7e(HL4), (C)0.04414.9

2260.2e(HL4), (D)0.02783.81537.9e(HL4), (E)0.03053.3

1441.6e(FL4), (A)

0.0754

5.8

22103

e(FL4), (B)0.0683

5.5

21

93.3

e(FL4), (C)0.0478

4.3

1965.3e(FL4), (D)0.03354.3

1645.8e(FL4), (E)0.03193.3

1243.5Lysozyme

0.0104

1.5

4.7

14.3

Table 2: Summary of SEC-MALS analyses ofePN-Block homooligomers
ePN-Block homooligomers
MolecularMultimers
Samples and peakmass [kDa]e(HL4), (A) Peak 1

129

Pentamere(HL4), (B) Peak 1

98.8

Tetramere(HL4), (C) Peak 2

73.5

Trimere(HL4), (D) Peak 1

48.6

Dimere(HL4), (E) Peak 2

48.8

Dimere(FL4), (A) Peak 1

135

Pentamere(FL4), (B) Peak 1

104

Tetramere(FL4), (C) Peak 1

69.6

Trimere(FL4), (D) Peak 1

47.6

Dimere(FL4), (E) Peak 2

48.3

Dimer

References[1] N. Kobayashi and R. Arai, Curr. Opin. Biotech., 46,57 (2017).
[2] R. Arai et al., J. Phys. Chem. B, 116, 6789 (2012).
[3] S. C. Patel et al., Protein Sci., 18, 1388 (2009).
[4] M. H. Hecht et al., Protein Sci., 13, 1711 (2004).
[5] N. Kobayashi et al., J. Am. Chem. Soc., 137, 11285(2015).
[6] R. Arai et al., Protein Eng., 14, 529 (2001).
[7] R. Arai et al., Proteins, 57, 829 (2004).
* rarai@shinshu-u.ac.jp