Thermal unfolding process of dihydrolipoamide dehydrogenase studied by fluorescence spectroscopy.
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 degrees 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.
Nishimoto
Etsuko
E
Institute of Biophysics and Institute of Protein Chemical Engineering, Faculty of Agriculture, Graduate School of Kyushu University, Hakozaki, Fukuoka 812-8581.
Aso
Yoichi
Y
Koga
Toshiaki
T
Yamashita
Shoji
S
D008058:Dihydrolipoamide Dehydrogenase / Q000737:chemistry*; D005454:Fluorescence Polarization; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006358:Hot Temperature; D008958:Models, Molecular; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D008058:Dihydrolipoamide Dehydrogenase