Photon Factory Activity Report 2005 #23 Part B (2006)
Biological Science

15A/2002G336

SAXS study on the halophilic ferredoxin. Salt concentration dependence
232Haripalsingh SONAWAT 1, Jinsong LI , Masaji SHINJO , Yoshitaka MATSUMURA2 , Satoshi NAKAMURA ,Toshiharu HASE4 and Hiroshi KIHARA2
1
Dept. of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai India
2Dept. of Physics, Kansai Medical University, 18-89 Uyama-Higashi, Hirakata 573-1136, Japan
3Dept. of Bioengineering, Tokyo Institute of Technol., 4259 Nagatsuda, Yokohama 226-8501, Japan4Institute of Protein Research, Osaka University, 3-2 Yamadaoka, Suita 565-0871, Japan

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Introduction
Ferredoxin is a group iron-sulfur protein that was foundin almost all living cultures. Although the ferredoxins
have wide range diversity, from primary sequence to thecomposition of amino acid, they share similar structure
and biological function. Halophilic archaea aremicroorganisms that require 3-5 mol of salt for a proper
life style that are lethal for common organisms.
Ferredoxin from Halobactrium salinrum is composed by
128 amino acids with a Fe2-S2 center and it works as theelectron carrier in the decarboxylation of a-ketoacids [1].
The sequence and structure of this halophilic ferredoxin isvery similar with plant type ferredoxin. However, thehalophilic ferredoxin has an additional domain at the Nterminus that includes large amount of negative charges.
This suggests the negative charge play an important roleto stabilize the halophilic ferredoxin in the high salt
concentration.
Here, we report the conformational study on halophilic
ferredoxin in the presence of various concentration ofsodium chloride by small angle x-ray scattering.

Data demonstrates; (1) mesophilic ferredoxin frommaize is compact with Rg of 14.1 Å both in 0M and in
4.26M NaCl. (2) Both halophilic ferredoxin fromHalobacterium salinarum and Haloarcula japonica arecompact in 4.26M NaCl, while they are unfolded in 0M
NaCl buffer. The molecule size is proportional tomolecule weight as far as they are globular except
halophilic ferredoxins in 4.26M NaCl, as shown in Fig 1.

Result
The halophilic ferredoxin is keeping compact when
[NaCl] higher than 1M, judging from radius of gyration(Rg) and Kratky plot. It is interesting that the Rg of theprotein, 20 Å, is larger than we predicted from its
molecule weight and that calculated based on the protein
data bank. At low salt concentration, the protein wasexpanded to 27 Å. This value is similar with the Rg of thechemically denatured state. It indicates halophilic
ferredoxin is totally expanded at low salt concentration.
In addition, we measured other two kinds of ferredoxinsin the extremely high salt concentration and at thephysiological condition. Maize ferredoxin is distributeddifferentially in mesophyll and bundle-sheath cells frommaize [2]. The Kratky analysis indicated the Maize
ferredoxin is kept compact both in the extremely high salt
and in the physiological condition. Gunier-plot fitting
analysis gave the Rg of maize ferredoxin 14.2 Å at thenative state.
Another halophilic ferredoxin from Haloarcula
japonica strain TR1 is a 32 kD protein [3]. Guinier-plot
fitting analysis gave the Rg of the protein in 4.26M NaClas 28 Å, while that in 0M NaCl buffer was 56 Å.

Using this linear relation, the molecular weight of thehalophilic ferredoxin and Haloarcula japonica, predictingfrom its molecule size, is very close to the double of theirmolecular weight, about 26kD, as shown in table 1. Thisimplies the proteins mainly take dimer in solution.

Users' Report

Table 1 Calculated molecular weight from Rg

Sample Mwa)
HAT 32KDa
Maize 10.5Kda
Halo 14.6KDaa)

Rgb)
20.9
14.5
16.1

Rg c)
2613.8
20.6Mwd)
60.5KDa
9KDa
30.1KDathe molecular weight was calculated from sequenceb)c)

the Rg was obtained from the molecular weightthe Rg was obtained from the experimental resultd)

The molecular weight was calculated from Rg

References[1] Bandyopadhyay et al., Biophys J. 79 (2000) 501-10.
[2] Matsumura, T., et al., Plant Physiol 119 (1999) 481-8[3] Matsuo T, et al., Biometals. 14 (2001) 135-42*kihara@makino.kmu.ac.jp