Photon Factory Activity Report 2019 #37 (2020)

BL-10C/2018G130

Molecular evolution of myoglobin upon land-to-sea transition of whales traced by thestructure factor of small-angle X-ray scattering
Hiroshi IMAMURA1,*, Tomonari SUMI2, and Yasuhiro ISOGAI3
Department of Applied Chemistry, College of Life Sciences, Ritsumeikan University, 1-1-1
Nojihigashi, Kusatsu, Shiga 525-8577, Japan
2
Research Institute for Interdisciplinary Science, Okayama University, 3-1-1 Tsushima-Naka,Kita-ku, Okayama 700-8530, Japan.
3
Department of Pharmaceutical Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu,Toyama 939-0398, Japan
11 Introduction
Extant whales are capable of storing much oxygen in theirmuscles to dive deeply. The concentration of an oxygenbinding protein, myoglobin (Mb), in aquatic mammals is~10 times higher than in terrestrial mammals [1]. Aquatic
mammal Mb evolved so that it should not aggregate at highconcentrations. That lets us ask what kind of molecularmechanisms underlies for that. To address this, weresurrected ancestral whale Mbs based on existing globin
sequences (Fig. 1) [1]. Two resurrected Mbs (aMbWb′ andaMbWb) are the common ancestors of the toothed andbaleen whale Mbs, which are closely related to Mb of
Basilosaurus in the sea ~40 million years ago (Mya). Theolder Mb, aMbWp, is a further common ancestor of whaleand hippopotamus Mbs, which would correspond to Mb ofa quadruped terrestrial animal (Pakicetus) ~50 Mya.
In the present study, to track the molecular evolution ofwhale Mb, we determined the self-molecular interaction ofthese ancestral Mbs and the extant ~3000 m-diving sperm
whale myoglobin (swMb) by using a small-angle X-rayscattering (SAXS). We analyzed the structure factors S(q),i.e., inter-particle interference, derived from the radialdistribution of the protein molecules in solution; theprotein-self interaction governs the radial distribution. We
determined a second virial coefficient (A2), an indicator ofrepulsion (A2 > 0) and attraction (A2 < 0) of molecules insolution.
aMbWp

aMbWb’

aMbWb

swMb
  

Early Eocene epoch~50 MyaPakicetus  PDB:5YCG
pI = 7.0

PDB:5YCH
pI = 8.0

PDB:5YCIpI = 8.2

Middleddle Eocen
Eocene epoch

PDB:5YCE
pI = 8.2

Present0 Mya~40silos
Basilosaurus

Sperm whale

Hippopotamus

Fig. 1: Molecular evolution of whale Mb. The illustrationsof animals are credited to Satoshi Kawasaki(https://paleontology.sakura.ne.jp).

2 Experiment
Mb proteins, aMbWp, aMbWb′, aMbWb, and swMb,
were synthesized by using E. coli and purified aspreviously described [1]. The Mb solutions were dialyzed
against a 2 mM HEPES-NaOH buffer solution (pH 6.8) at
4 °C for one day. The dialyzed Mb solutions wereconcentrated to ~3–5 mM, and then centrifuged. The Mbsolutions were diluted to the desired concentrations at a pHof 6.9 ± 0.1, and irradiated with X-ray wavelength λ = 0.15
nm for 2 s in a cell with quartz windows using a sampleflow system (~14.5 μL/min) at 20 ± 0.1 °C. The SAXS
experiment was performed at the beamline BL-10C. Thecamera length of 1 m was calibrated by use of a scattering
pattern of silver behenate. X-ray intensities were recorded
by a PILATUS3 2 M detector (DECTRIS Ltd.,
Switzerland). A total of 30 images were collected for eachcondition, and the circular 1D averaging of the images wasperformed with the program Nika [2]. The scatteringparameter q = |q| = 4πsinθ/λ, where q is the scatteringvector and 2θ is the X-ray scattering angle. The scattering
intensity of the protein was converted to the absolutescattering intensity (I(q)) by use of water scattering as thestandard [3].
3 Results and Discussion
I(q) was extrapolated to the scattering intensity at q = 0,I(0), which is related to A2 as
I(0) = kMcpS(0) (1)andS(0) = 1 / (1 + 2A2Mcp) (2)where M is the molecular weight of the protein, cp is theprotein concentration, the k value is equal to v2(ρm ρsolv)2/NA, and S(0) is S(q) at q = 0. NA is Avogadro’s
number, v is the partial specific volume of the protein (here,0.7425 cm3 g  1), and ρm  ρsolv is the electron density
difference between the protein and the solvent (2.8 × 1010cm2) [4].
SAXS intensities in the small q region were depressed
for all the Mbs by an increase of Mb concentration,
indicating repulsive self-interaction between Mb
molecules in solution. SAXS profiles of aMbWp in Fig.
2(a) were dependent on the Mb concentration to less extent
than those of swMb in Fig. 2(b). Fig. 2(c) shows that A2 ofMbs, with the oceanic adaptation of whales, increased, i.e.,
increased repulsion via the molecular evolution. Since thePhoton Factory Activity Report 2019 #37 (2020)

net charge increase converges in the first half of evolution(aMbWp to aMbWb), the increase of repulsion in thesecond half of evolution (aMbWb to swMb) would not besatisfactorily explained by the change in the net charge. On
the other hand, the structural stability was enhanced in thelatter evolution, suggesting a correlation with it (see thereference [1]). For further study, we will progress SAXS
studies on the other terrestrial and aquatic mammal Mbs.
(a)

(b)

2

aMbWp

10 8c / mg mL
1.1
2.3
3.4
4.5
5.7
6.88.2
9.1
10111427

642-1-1 -1/ [cm (mg mL ) ]

-2-3

10-1

86I(q) c

42-4

10

70.01

-1

22-2SWMb

10 86c / mg mL
0.45
0.89
1.2
2.4
3.6
4.8
6.0
7.2
8.6
9.5
1142-3108642-4

34 5 6 7

0.1

2

34 5

10

70.01

-1

1214293

4 5 6 7

0.1

23

4 5-1

q/Å

(c)

2-1

q/Å

Repulsive

63

54-4-2A2 / (10 cm mol g )73210aMbWp

aMbWb'aMbWb

SWMb

Fig. 2: SAXS analysis of the self-interaction of ancestraland extant Mbs. Concentration-dependence of SAXS
profiles of (a) aMbWp and (b) swMb. Change in repulsive
interaction between Mb molecules is represented by (c) A2.
Acknowledgement
We thank Satoshi Kawasaki for kind permission to use
his illustrations of animals. This work was supported inpart by Public Interest Incorporated Foundation of Institutefor Chemical Fibers, Japan (to H.I.).
References[1] Isogai et al., Sci. Rep. 8, 16883 (2018)[2] Ilavsky, J. Appl. Crystallogr. 45, 324 (2012).
[3] Orthaber et al., J. Appl. Crystallogr. 33, 218 (2000).
[4] Mylonas and Svergun, J. Appl. Crystallogr. 40, S245
(2007).
* himamura@fc.ritsumei.ac.jp