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http://dx.doi.org/10.6564/JKMRS.2013.17.2.067

Improved Purification of Thermophilic FoF1-ATP Synthase c-Subunit Rings and Solid-State NMR Characterization of Them in Different Lipid Membranes  

Bak, Suyeon (Department of Biophysics and Chemical Biology, Seoul National University)
Kang, Su-Jin (Department of Biophysics and Chemical Biology, Seoul National University)
Suzuki, Toshiharu (Chemical Resources Laboratory, Tokyo Institute of Technology)
Yoshida, Masasuke (Chemical Resources Laboratory, Tokyo Institute of Technology)
Fujiwara, Toshimichi (Institute for Protein Research, Osaka University)
Akutsu, Hideo (Department of Biophysics and Chemical Biology, Seoul National University)
Publication Information
Journal of the Korean Magnetic Resonance Society / v.17, no.2, 2013 , pp. 67-75 More about this Journal
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 $F_oc$-ring ($TF_oc$-ring) for NMR measurements. The expression of $TF_oF_1$ 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 $TF_oF_1$. $TF_oc$-rings were purified from $TF_oF_1$ according to a reported method. We modified purification procedures to improve purity and yield of $TF_oc$. 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 $TF_oc$-rings were reconstituted into two kinds of lipid bilayers, namely, saturated and unsaturated lipid ones. Then, we have compared characteristics of the $TF_oc$-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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