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Journal of Biochemistry Advance Access originally published online on July 21, 2006
Journal of Biochemistry 2006 140(3):349-357; doi:10.1093/jb/mvj156
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© 2006 The Japanese Biochemical Society.

ARTICLE

Thermal Unfolding Process of Dihydrolipoamide Dehydrogenase Studied by Fluorescence Spectroscopy

Etsuko Nishimoto1, Yoichi Aso2, Toshiaki Koga3 and Shoji Yamashita1,*

1 Institute of Biophysics and 2 Institute of Protein Chemical Engineering, Faculty of Agriculture, Graduate School of Kyushu University, Hakozaki, Fukuoka 812-8581; and 3 National Institute of Advanced Industrial Science and Technology, Tosu, Saga 841-0052

* To whom correspondence should be addressed. Tel/Fax: +81-92-642-4425, E-mail: yamashita{at}brs.kyushu-u.ac.jp

The thermal unfolding pathway for dihydrolipoamide dehydrogenase (LipDH) isolated from Bacillus stearothermophilus was investigated focusing on the transient intermediate state characterized through time-resolved fluorescence studies. The decrease in ellipticity in the far UV region in the CD spectrum, the fluorescence spectral change of Trp-91 and FAD, and the thermal enzymatic inactivation curve consistently demonstrated that LipDH unfolded irreversibly on heat treatment at higher than 65°C. LipDH took a transient intermediate state during the thermal unfolding process which could refold back into the native state. In this state, the internal rotation of FAD was activated in the polypeptide cage and correspondingly LipDH showed a peculiar conformation. The transient intermediate state of LipDH characterized in time-resolved fluorescence depolarization studies showed very similar properties to the molten-globule state, which has been confirmed in many studies on protein folding.


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