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

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.