Photon Factory Activity Report 2019 #37 (2020)

BL-6A, BL-10C, BL-15A2/2017G566

Single-Pair SAXS for Analyzing a Conformational Ensemble of Heavy Atom-Labeled
Small Molecules
Yuya TAGUCHI Tomohide SAIO and Daisuke Kohda
Medical Institute of Bioregulation,
Kyushu University,
3-1-1Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan
Faculty of Science, Hokkaido University, Sapporo, Hokkaido 060-0810, Japan
121,*121 Introduction
It is difficult to obtain unbiased spatial information on
the dynamic conformational ensemble of a molecule insolution. There are several experimental methods, but they
have inherent problems in achieving this demanding task.
One promising approach is small-angle X-ray scattering(SAXS) by molecules in a liquid solution. A scattering
curve, I(q), obtained from a SAXS experiment can beconverted to a pair distribution function, P(r). The function
P(r) describes the distribution of distances between all
pairs of atoms in a molecule (Fig. 1, upper panel). If we
introduce a pair of strong scatterers as probes into amolecule and selectively measure the SAXS profiles fromthe two scatterers, then the P(r) function will describe thespatial distribution of the inter-probe distance (Fig. 1,lower panel). The background SAXS profile originatingfrom the other atoms in the molecule must be suppressed.
Gold nanocrystals have been successfully used as strong
scatterers . Gold nanocrystals are attached via a gold-thiol
linkage to the terminal and middle positions of doublestranded (ds) DNA fragments, ranging from 10 to 35 bp.
The background scattering from the DNA part is
suppressed by the combination with scattering data fromsingly labeled and unlabeled DNA samples, according to
the equation I (q) - I (q) - I (q) + I (q). Note that there are
two different scattering data corresponding to two singly
labeled species. The typical distance ranges are limited
between 50 and 150 Å in the gold-labeled DNA
experiments. The large size (diameter of 12-14 Å) and thesize distribution of the gold nanocrystals, and the necessityof the linker for immobilization, reduce the usefulness formonitoring the dynamics within a small molecule. Heavy
single atoms directly attached to a target molecule withouta linker would be preferable in this respect but have notbeen experimentally assessed so far.

concentration of 29 mM. The profiles were added andsubtracted according to the equation I (q) – 2 * I (q) + I (q),to extract the X-ray scattering interference pattern between
two iodine atoms connected by a flexible PEG chain (Fig.
2).
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11’02 Experiment
Small-angle X-ray scattering was measured at thebeamline BL6A of the Photon Factory. The sample volume
was 40 µL, and a microflow cell was used to reduce X-rayirradiation damage. The X-ray wavelength was set at 1.5 Åand the sample-detector distance was 1.0 m. Data werecollected on a PILATUS 1M detector in twenty 20-second
exposures per sample. Three scattering profiles werecollected from samples of unlabeled, singly labeled, anddoubly labeled PEGs dissolved in DMSO at a finalFig. 1. TOC summary of the single-pair SAXS
measurement
We used a modified version of the MATLAB programused in the study on the gold-nanocrystal labeled DNA
fragments1. The program is available in the supplementary
information of ref 2.
3 Results and Discussion
We present a feasibility demonstration to show that thedistance distribution between two heavy atoms in smallorganic molecules in the range of 10 - 30 Å can beexperimentally obtained . Iodine was chosen as a strong Xray scatterer and covalently attached to the ends of aflexible polyethylene glycol [PEG, -(CH -CH -O-) ]chain(Fig. 2). Since the scattering power of a single iodine atom
is much smaller than that of a gold nanoparticle (whichconsists of 78 gold atoms), the feasibility must beexamined experimentally. We refer to the compounds inFig. 2 as I -PEGn, I -PEGn, and I -PEGn, where I , I , and I
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Photon Factory Activity Report 2019 #37 (2020)

denote the number of iodine atoms, and n denotes thenumber of ethylene glycol units (n = 3, 5, 9). Note that IPEGn-H and H-PEGn-I are identical due to the C2
symmetry, and referred to simply as I -PEGn.
1Fig. 3 SAXS derived I-I distance distributions of thedouble-iodine labeled PEG chains.

Fig. 2. Chemical structures of the iodine-labeled PEGs.
The maximum iodine-iodine (I-I) distances are 25.1 Åfor I -PEG , 31.6 Å for I -PEG , and 45.2 Å for I -PEG.
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The distance distribution between the two iodine atoms
was obtained by decomposing the interference pattern intoa linear combination of interference basis functions
between two iodine atoms, positioned at various distancesfrom 0 to 200 Å with 1 Å intervals (Fig. 3). The scattering
data in the range of 0.008 Å < S < 0.064 Å were used forinput. The decomposition was performed by the maximum
entropy procedure coded in MATLAB.
Our proof-of-concept SAXS experiment was proved
useful in extracting distance distributions in the range of10 – 30 Å. The present single-pair (of heavy atoms) SAXS
experiments widen the applicability of SAXS
interferometry from ds DNA fragments to flexible smallorganic compounds in the physical and chemical fields.
We also expect to obtain new insights into the spatial
dynamics of folded and IDP (intrinsically disorderedpolypeptide)-state proteins in aqueous solutions forbiological applications.
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AcknowledgementWe thank Drs. Hiroshi Nonaka and Shinsuke Sando(Graduate School of Engineering, The University ofTokyo) for the chemical synthesis of N-succinimidyl-4iodobenzoate. We thank Dr. Nobutaka Shimizu atbeamlines BL-6A, BL-10C, and BL-15A2 of the Photon
Factory, and Dr. Takaaki Hikima at beamline BL-45XU,
for assistance with the SAXS measurements.

References[1] Mathew-Fenn RS, Das R, Silverman JA, Walker PA,Harbury PA. A molecular ruler for measuring
quantitative distance distributions. PLoS One. 3:e3229(2008).
[2] Taguchi Y, Saio T, Kohda D. Distance Distribution
between Two Iodine Atoms Derived from Small-Angle
X-ray Scattering Interferometry for Analyzing a
Conformational Ensemble of Heavy Atom-Labeled
Small Molecules. J. Phys. Chem. Lett. 11: 5451-5456(2020).
* kohda@bioreg.kyushu-u.ac.jp