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JKMRS Volume 17, No. 2, 67-75 : Improved Purification of Therm...
2013년 12월 20일 / 조회수: 772

Improved Purification of Thermophilic FoF1-ATP Synthase

c-Subunit Rings and Solid-State NMR Characterization of Them

in Different Lipid Membranes

 

 

Suyeon Bak1, Su-Jin Kang1, Toshiharu Suzuki3,4, Masasuke Yoshida3,4, Toshimichi Fujiwara2, and Hideo Akutsu1,2*

 

1Department of Biophysics and Chemical Biology, Seoul National University, Kwanak-Gu, Seoul 151-742, Republic of Korea,

2Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita 565-0871, Japan,

3Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259,

Yokohama 226-8503, Japan,

4Department of Molecular Bioscience, Kyoto Sangyo University, Kamigamo-Motoyama, Kyoto 603-8555, Japan.

 

 

Received Jul 17, 2013; Revised Aug 7, 2013; Accepted Aug 14, 2013

 


Abstract   ATP synthase produces ATP, a major energy source for metabolic processes in organisms, from ADP and inorganic phosphate in cellular membranes. ATP synthase is known as a rotary motor, in which the c-subunit ring functions as a rotor. In this work, we have tried to develop a more general preparation procedure of thermophilic Foc-ring (TFoc-ring) for NMR measurements. The expression of TFoF1 is easily affected by various experimental conditions such as temperature, shape and size of a flask, a volume of medium, and shaking rate of an incubator. Accordingly, we have tried to optimize the expression conditions of TFoF1. TFoc-rings were purified from TFoF1 according to a reported method. We modified purification procedures to improve purity and yield of TFoc. On top of them, we found a new combination of detergents for the purification at anion-exchange column chromatography. To examine the effect of lipid environments on the structure, the TFoc-rings were reconstituted into two kinds of lipid bilayers, namely, saturated and unsaturated lipid ones. Then, we have compared characteristics of the TFoc-ring structures in these membranes with solid-state NMR.

 

Keywords   motor protein, membrane protein, ring His-tag, detergent, CP/MAS NMR
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