Photon Factory Activity Report 2022 #40 (2023)

BL-10C/2020G003, 2022G003

Novel function of pigeon iron–sulfur protein for the magnetoreception
predicted by SAXS analysis with a permanent magnetic device
Shigeki ARAI1*, Rumi SHIMIZU1, Motoyasu ADACHI1, Mitsuhiro HIRAI2
1National Institutes for Quantum Science and Technology,
4-9-1 Anagwa, Inage, Chiba-city, Chiba, 263-8555, Japan
2Graduate School of Science and Technology, Gunma University, 4-2 Aramaki, Maebashi, Gunma
371-8510, Japan
1 Introduction
Magnetoreception, which is a sense to perceive themagnetic field information, is used for many animal
behaviors (migratory, homing, feeding, breeding, etc.) [1].
Cryptochrome (CRY) in the retinal cell of some animal
species can act as a quantum sensor to detect the angle of
the geomagnetic field [2,3]. However, partner proteins for
CRY, such as CRY receptors that transmit magnetic
information from CRY to perception and scaffold proteinsthat assist the magnetoreception function of CRY
molecules, have not yet been identified. If CRY molecules
are not anchored in retinal cells by assistant substances,
CRY molecules will be randomly oriented, rotated anddiffused, and the detected angle of the magnetic field will
differ for each CRY molecule. In such situations, the angleof the magnetic field line would not be perceived correctly.
On the other hand, Qin et al. found that the iron–sulfur(Fe–S) cluster assembly homolog 1 of pigeon (clISCA1)interacts with CRY (clCRY4) and the clCRY4/clISCA1

Fig. 1. Time course (0 – 60 min) of SAXS curves of
6.9 mg/mL clISCA1 solution with applying the external
static magnetic field. (a) the condition A with 180 mT.
(b) the condition B with –40 mT.

complex tends to be orientated by weak magnetic fields (40μT–1 mT) [4]. These results suggest that the ISCA1 may
be one of the candidates of CRY partners. In the previousstudy, we clarified that clISCA1 molecules in a clISCA1solution form columnar oligomers under the existence ofFe-S clusters, and these columnar oligomers in a 29.3
mg/mL clISCA1 solution elongate due to the self-assembly
induced by applying the static magnetic field (<1 T) [5,6].
Recently, we further investigated the magnetic response ofthe molecular behavior of clISCA1 at lower magneticfields (<180 mT) and a lower clISCA1 concentration (6.9mg/mL). In this report, we summarize our observations
using SAXS and previous studies. A novel function ofclISCA1 for the magnetoreception can be presumed fromthis summary.
2 Experiment
The construction of clISCA1 was described previously[5]. Approximately a 40 μL sample solution containing 6.9
mg/mL clISCA1, 20 mM Tris-HCl buffer (pH 8), 150 mM
NaCl and 10 mM 3-mercapto-1,2-propanediol was usedfor SAXS measurements. The wavelength used was 1.55
Å. The sample-to-detector distance was 3 m. The clISCA1solution was injected into a sample cell with a 2 mm pathlength. A sample cell was inserted into a periodic Nd–Fe–
B permanent magnetic circuit set in the BL-10C [7] asshown in Fig.5a of ref. [5] and Fig.2a of ref. [6]. Two
different conditions of the magnetic field (condition A andcondition B) were generated in the clISCA1 solution bychanging the position of a sample cell in a magnetic circuitand the gap (1~2.5 cm) of a magnetic circuit [7]. For thecondition A, the difference of the magnetic flux densities
in the range from 160 to 180 mT was generated in a sample
cell, and the magnetic flux density at beam center wasadjusted to 180 mT. For the condition B, the difference ofthe magnetic flux densities in the range from –20 to 100
mT was generated in a sample cell, and the magnetic flux
density at the beam center was adjusted to –40 mT. SAXS
data during the application of the external static magnetic
field for 60 min were collected using a PILATUS3 2M
detector (Detectris) at 20 ˚C. X-ray exposure time to collect
one SAXS curve was 1min. Buffer scattering wassubtracted from each curve to yield the sample scattering
curves I(q)s using the program SAngler ver.2.1.64 [8].
Since clISCA1 forms columnar oligomers, the averagecross-sectional radius of gyration (Rc) was evaluated using
the program SCÅTTER ver.4 with equations (1) and (2) [9].

Photon Factory Activity Report 2022 #40 (2023)

𝐼(𝑞) = 𝐼𝑐 (𝑞)⁄𝑞(1)𝐼𝑐 (𝑞) = 𝐼𝑐 (0)𝑒𝑥𝑝(−𝑅𝑐2 𝑞2⁄2)(2)

Ic(q) denotes the scattering curve of the cross-section of theparticle. q is the magnitude of the scattering vector. The
Guinier region of Ic(q) can be approximated by theequation (2). The Rc and Ic(0) values can be evaluated froma linear fit to the cross-sectional Guinier plot (ln{q • I(q)}vs. q2 plot). I(q)s were further converted to the distance
distribution function P(r)s by Fourier transform using theprogram GNOM in ATSAS ver.3.1.3 [11]. The maximum
diameter (Dmax) of the clISCA1 columnar oligomer wasestimated from the P(r) function satisfying the condition
P(r) = 0 for r > Dmax.
3 Results and Discussion
Fig.1a and 1b show the time course of SAXS curves ofclISCA1 for the conditions A and B, respectively. In both
Fig.1a and 1b, the scattering intensities at q < 0.01 Å−1 ofI(q)s increased with the retention time of the clISCA1solutions in the magnet. Since these changes in I(q)s was
not due to protein aggregation by radiation damage [3,4],
the result indicated that the oligomerization of clISCA1
molecules was magnetically induced. Fig.2a and 2b show
P(r) functions obtained from SAXS curves shown inFig.1a and 1b, respectively. The profiles of P(r)s in Fig.2aand 2b were changed from a double peak (0 min) to a tailedpeak (60 min) according to retention time of the clISCA1solution in the magnet, suggesting that the particles in theclISCA1 solution changed from a dumbbell shape(probably dimer or more) to a columnar shape. Dmax of theFig. 2. P(r) functions of clISCA1 obtained by Fouriertransform from I(q)s of (a) Fig. 1a and (b) Fig.1b.

clISCA1 oligomer increased from 73 to 113 Å for thecondition A and from 73 to 108 Å for the condition B(Fig.3). Conversely, Rc during 60 min of retention time in
the magnetic was almost constant (17±2 Å for thecondition A and 13±1 Å for the condition B) as shown in
Fig.3. These results indicated that the cross-section
diameter of the columnar clISCA1 oligomer does notchange, but the columnar oligomer’s long axis elongatesby applying the external static magnetic field [5].
With a retention time of 0 min in the magnet (under thenatural geomagnetic condition), Dmax measured in thisstudy (73 Å) was smaller than that measured using 29.3
mg/mL clISCA1 solution in the previous study (158 Å) [5].
This result indicates that the length of the clISCA1columnar oligomer depends on both the magnetic field
condition and the clISCA1 concentration. However, thetendency to elongate magnetically the clISCA1 columnar
oligomer shown here was very similar to the result in theprevious study [5,6]. Therefore, the present results
demonstrate that the translational diffusion andoligomerization of clISCA1 are magnetically responsive
under conditions closer to the natural environment than inprevious studies [5,6].
Although it is technically difficult to clarify the role ofthemagnetic
orientational
propertyofclCRY4/clISCA1 oligomer and the magnetic responseof clISCA1 in the vicinity of geomagnetic fields (<0.1 mT),
clISCA1 certainly responds to the external magnetic field.
Since the clISCA1 oligomer binds two types of Fe–S
clusters ([2Fe–2S] and [3Fe–4S] cluster) and mononuclear
Fe atoms [12,13], the magnetism of clISCA1 oligomer
should be regulated by the synergistic effect ofFig. 3. Time course of Dmax and Rc of the clISCA1
columnar oligomer in the magnetic circuit. In the upperfigure, open circles and open triangles indicate Dmax ofthe condition A and the condition B, respectively.
Closed circles and closed triangles indicate Rc of thecondition A and the condition B, respectively. The
lower figure shows the schematic drawing of theclISCA1 oligomer connected by Fe–S clusters.

Photon Factory Activity Report 2022 #40 (2023)

mononuclear Fe atoms, [3Fe–4S] / [2Fe–2S] mixing
binding and the redox state of Fe–S clusters [5,12,13]. For
example, [2Fe–2S]1+ (containing one Fe(II) and one
Fe(III)) and [3Fe–4S]0 (containing one Fe(II) and twoFe(III)s) become paramagnetic states. Conversely, [2Fe–2S]2+ (containing two Fe(III)s) and [3Fe–4S]1+ (containingthree Fe(III)s) become a diamagnetic state [14].
When the external static magnetic field is applied to asample solution, the ferromagnetic, ferrimagnetic,superparamagnetic, paramagnetic, and antiferromagnetic
particles in a solution gather at a high magnetic intensity
location. Conversely, the diamagnetic particles gather at alow magnetic intensity location. Therefore, the clISCA1
oligomers formed at low and high magnetic field
intensities would have different magnetic states, namely
diamagnetic and nondiamagnetic states, respectively. Morespecifically, the oligomerization ability of clISCA1 in thenondiamagnetic state may be improved at high magnetic
flux density locations, such as high-latitude regions of theEarth (Fig.4).
Moreover, clCRY4 may recognize the nondiamagnetic
clISCA1 oligomers, since the clCRY4/clISCA1 complex
behaves like a ferrimagnetic material [4] and the clISCA1
oligomer bound to both Fe–S clusters and mononuclear Fe

atoms behaves as a paramagnetic substance averagely at
300 K [12]. Taken together, the amount of clCRY4molecules anchored on the clISCA1 oligomer will increaseat high-latitude regions, which may increase the amount offunctional clCRY4 molecules as compass magnets. As aresult, the signal intensity of the magnetic informationtransmitted from clCRY4 to perception may be changed
depending on the geomagnetic field intensity. If clISCA1
acts as a scaffold for clCRY4, pigeon may be able toperceive not only the angle of the magnetic field line but
also the magnetic flux density reflecting the latitude.
Acknowledgement
This work was supported by JSPS KAKENHI [grantnumbers 18K06174 and 21K06093] and MEXT Quantum
Leap Flagship Program (MEXT Q-LEAP) [grant number
JPMXS0120330644]. Synchrotron radiation experiments
were performed at the BL-10C of Photon Factory under theapproval of the Photon Factory Program Advisory
Committee (Proposal Nos. 2020G003 and 2022G003).

Fig. 4. A schematic drawing of the predicted mechanism of clCRY4/clISCA1 complexation in the geomagnetic field,assuming that the clISCA1 molecules are in the nondiamagnetic states. For example, the magnetic flux densities at theEarth’s surface of the geomagnetic north and south poles are 65 – 67 μT, which are approximately three times higher
than that near the equator (22 μT) [15–17]. The nondiamagnetic clISCA1 may polymerize at high-latitude regions andbind to clCRY4 molecules.

Photon Factory Activity Report 2022 #40 (2023)

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mlResearch Achievements
https://www.qst.go.jp/site/press/20220511.html* arai.shigeki@qst.go.jp