Photon Factory Activity Report 2017 #35 (2018)

BL-10C/2016G032

Small angle X-ray scattering of an artificial four helix bundle protein
Hiroshi IMAMURA1 and Yasuhiro ISOGAI2,*
Department of Applied Chemistry, College of Life Sciences, Ritsumeikan University, 1-1-1
Nojihigashi, Kusatsu, Shiga 525-8577, Japan2
Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama,939-0398, Japan
12 Experiment
The amino acid sequence of the protein, bHH is
HMLKKLREEHLKLLEEFKKLLEEHLKWLEGGGGG
GGGELLKLHEELLKKFEELLKLHEERLKKL, which
was synthesized by using E. coli and purified aspreviously described [2]. The SAXS experiment wasperformed at the beamline BL-10C. The X-ray
wavelength, λ, was 0.1488 nm; the camera length was1049 mm, which was calibrated by use of a scattering
pattern of silver behenate. X-ray intensities were recorded
by PILATUS3 2M (DECTRIS Ltd., Switzerland). bHH
was dialyzed against the aqueous solution with 10 mM
HEPES-NaOH and 50 mM NaCl (pH 7.2). The protein
concentration was 2.8 mg/mL. After centrifugation, thesupernatant solution was measured. 150 images werecollected; an image was recorded for 2 sec of theexposure time. The temperature was controlled to be
10 °C. The circular 1D average of the image wasperformed by the program Nika [3]. The 1D data wereaveraged; damage by radiation was negligibly small. Thescattering parameter q is defined as q = 4πsinθ/λ, where2θ is the scattering angle of X-rays.
3 Results and Discussion
Fig. 1 shows the SAXS profile of the bHH protein.
Guinier analysis of the profile in the q region of 0.016–0.045 Å–1 gave 25.5 ± 0.1 Å of the radius of gyration (Rg).
A fitting analysis using a theoretical scattering function

indicates that the cylinder [4] with a radius of 14.6 ± 0.3
Å and the length of 77.9 ± 0.8 Å was a good
approximation for the experimental scattering as shown inFig. 1. According to the previous study [2], the two
chains of bHH were associated, where the single chain ofbHH formed a helix–loop–helix structure: the bHH was afour helix bundled. The given radius (14.6 Å) of thecylinder model for bHH was in agreement with thatexpected; we estimated the radius to be ~14 Å using thecrystal structure of the other four helix bundle protein(PDB: 1M3W). On the other hand, the given length (77.9Å) was longer than that expected (<~50 Å); it is assumed
that a helical turn has a pitch of 5.4 Å consisting 3.6residues. This implies that the misaligned helix bundledmolecules or further associated molecules would becontaminating.
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2I(q) / a.u.

1 Introduction
Creating water-soluble proteins has been one of thechallenging issues in a protein design research because oflack of knowledge about a protein self-interaction and asolvation mechanism. We have developed a method forextracting the protein self-interaction using small angle
X-ray scattering (SAXS) and a liquid state theory [1]. To
extend the study, we have targeted natural proteins suchas lysozyme and artificial proteins. The former is fordetermining the self-interaction potential, by which thenatural proteins avoid the aggregation or precipitation.
The latter is for getting insights of what kinds of design
are effective for making the artificial proteins soluble; yet,concentrating the artificial proteins for analysis of theinteraction often resulted in precipitation so far.
On the other hand, characterizing solution structures of
the artificial proteins is beneficial for improving thestructure and the stability. SAXS analysis is preferredbecause it is often bottlenecking to crystallize proteins.
Accordingly, we have conducted SAXS measurement for
the artificial proteins designed by Isogai et al. (e.g., [2]).

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23

4-1q/ÅFig. 1: Small angle X-ray scattering profile of theartificial protein, bHH (black filled circles). Grayline
represents the best fit curve calculated based on a cylindermodel.
Acknowledgment
This work was supported in part by Public Interest
Incorporated Foundation of Institute for Chemical Fibers,
Japan (to H.I.).
References[1] Sumi et al., Phys. Chem. Chem. Phys. 16, 25492(2014)[2] Ishida et al., Biochemistry 43, 9823 (2004).
[3] Ilavsky, J. Appl. Crystallogr. 45, 324 (2012).
[4] Pedersen et al., Adv. Colloid Interface Sci. 70, 171(1997).
* yisogai@pu-toyama.ac.jp