Photon Factory Activity Report 2004 #22 Part B (2005)
Biological Science

15A/2003G317

Molecular properties for irreversible unfolding of aspergillopepsin II1
Takashi UCHIDA, Masaki KOJIMA*1, Hideshi INOUE1, Hiroshi KIHARA2, Kenji TAKAHASHI1
1Tokyo Univ.
of Pharm. and Life Sci., Hachioji, Tokyo 192-0392, Japan
2
Kansai Med. Univ., Hirakata, Osaka 573-1136, Japan

Introduction
Irreversible conformational change plays an essentialrole for controlling the physiological activity of proteins.
The study for the unfolding not only provides theinformation of molecular architecture, but is also
important to understand the control mechanism describedabove.
Aspergillopepsin II (aspergilloglutamic peptidase)
consists of two polypeptide chains, a light chain of 39
residues and a heavy chain of 173 residues, which arebound non-covalently to each other. This enzyme acts
under acidic condition, while it is unfolded irreversibly
above neutral pH accompanied with the dissociation of
the two polypeptide chains [1, 2]. It was suggested fromthe previous SAXS analyses that the dissociated heavy
chain is not completely coiled in the completely
unfolded state (pH 8.0 and above), and that the molecule
may have a core or residual structure. In the presentstudy, we further investigate the details of the structurein the alkaline-induced unfolded state by ab initio
modeling calculation.
Experimental
All experiments were performed at a sample-todetector length of 1.3 m and at room temperature with a
CCD-based X-ray detector. The exposure time was 30 s in
one measurement. The sample in the cuvette wasexchanged every three times. The data were corrected for
distortion of images, non-uniformity of sensitivity, andcontrast reduction for an X-ray image intensifier before
analyses [3].
The heavy chain was dissociated from the light chain at
pH 8.4, and purified by gel filtration. Sample solutions
were prepared at a protein concentration of 0.5 – 2.0
mg/ml.
ab inito modeling was performed with the program
DAMMIN [4] and GASBOR [5].
Results and Discussion
From the scattering profile, we first estimated thevarious parameters such as Rg and I(0). These parameters
were independent of sample concentration. Molecularweight deduced from I(0) value was consistent with thatof the heavy chain. Rg was estimated to be about 30 A.
Then we restored the low-resolution structure in theunfolded state by ab initio modeling. The restored
structure is expressed as an ensemble of small beads, andsatisfied the experimental scattering pattern. Fig. 1 shows
the result for the unfolded structure of the heavy chain.
Unlike the random coil, the molecule had an unisotropic

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Users' Reportshape, and bore a core inside itself. And the resultant
shape was almost the same irrespective of used algorithm.
We are trying to map this beads model into the atomiclevel high resolution structure.
References[1] M. Kojima et al., Biochemistry 39, 1364 (2000).
[2] M. Maeda et al., Biochem. Biophys. Res. Commun.
301, 745 (2003).
[3] K. Ito et al., PF Activity Report 18, 275 (2001).
[4] D. I. Svergun, Biophys. J. 76, 2879 (1999).
[5] D. I. Svergun et al. Biophys. J. 80, 2946 (2001).
* mkojima@ls.toyaku.ac.jp

Fig. 1. Restored structure of the heavy chain in theunfolded state. Beads in cyan and magenta correspondto the structure restored by DAMMIN and GASBOR,
respectively.