Improved Purification of Thermophilic FoF1-ATPSynthase c-Subunit Rings and Solid-State NMR Characterization of Them in Different Lipid Membranes
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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
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
참고문헌 (18)
- M. Yoshida, , Nat Rev Mol Cell Biol, 2, pp.669~677 (2001)
- P.D. Boyer, , Annu Rev Biochem, 66, pp.717~749 (1997)
- I.N. Watt, , Proc Natl Acad Sci U S A, 107, pp.16823~16827 (2010)
- S.P. Tsunoda, , Proc Natl Acad Sci U S A, 98, pp.898~902 (2001)
- H. Seelert, , Nature, 405, pp.418~419 (2000)
- L. Preiss, , PLoS Biol, 8, pp.e1000443~ (2010)
- N. Mitome, , Proc Natl Acad Sci U S A, 101, pp.12159~12164 (2004)
- T. Meier, , Science, 308, pp.659~662 (2005)
- D. Matthies, , J Mol Biol, 388, pp.611~618 (2009)
- H. Yagi, , J Biol Chem, 284, pp.2374~2382 (2009)
- T. Nakano, , J Mol Biol, 358, pp.132~144 (2006)
- A.M. Seddon, , Biochim Biophys Acta, 1666, pp.105~117 (2004)
- I. Yumen, , Protein Expr Purif, 82, pp.396~401 (2012)
- T. Suzuki, , J Biol Chem, 277, pp.13281~13285 (2002)
- K. Takegoshi, , Chem Phys Lett, 344, pp.631~637 (2001)
- T. D. Goddard, "SPARKY 3", University of California (2008)
- M. Kobayashi, , Biophys J, 94, pp.4339~4347 (2008)
- S. Leekumjorn, , J Phys Chem B, 111, pp.6026~6033 (2007)