Native Membrane Proteins vs. Yeast Recombinant: An Example: The Mitochondrial ADP/ATP Carrier
The mitochondrial ADP/ATP carrier (Ancp) has long been a paradigm for studies of the mitochondrial carrier family due to, among other properties, its natural abundance and the existence of specific inhibitors, namely, carboxyatractyloside (CATR) and bongk
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uction Major metabolic pathways occur within mitochondria, which is the primary site of ATP synthesis. It is isolated from the cytosol by two membranes (outer and inner) that are therefore important sites for the regulation of metabolic functions. The mitochondrial carrier family (MCF) members are integral membrane proteins that transport various metabolites through the mitochondrial inner membrane. Among them, the mitochondrial ADP/ATP carrier (Ancp) was the first to be identified around 40 years ago. Jean-Jacques Lacapère (ed.), Membrane Protein Structure Determination: Methods and Protocols, Methods in Molecular Biology, vol. 654, DOI 10.1007/978-1-60761-762-4_2, © Springer Science+Business Media, LLC 2010
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It plays a key role in the energetic cell metabolism because it exchanges ATP and ADP, respectively product and substrate of the mitochondrial ATP synthase. Ancp is the most abundant among MCF members (up to 10% of mitochondrial proteins in beef heart mitochondria) and has to cope with high nucleotide amounts in order to fulfill cell energetic requirements. It can be purified pretty easily in one or two steps from beef mitochondria in high amount and in crystallization compatible quality. However, the high-resolution structure of the beef Anc1p in complex with its inhibitor carboxyatractyloside (CATR) evidenced a monomeric organization (1). This was contradictory to many previously published results about different MCF members, of which Ancp shares functional and structural features and was shown to be organized as dimers (for a review see (2)). We hypothesized that protein preparation, and more precisely, the protein concentration step necessary to crystallization trials was responsible for Ancp dimer dissociation. Therefore, we used an engineered Ancp mutant (3) that forces a dimeric organization of the carrier throughout the purification and crystallization processes; we named it AA in this chapter. However, we previously showed that it was necessary to tag the yeast Ancp to get it highly purified (4). Consequently, the chimera was tagged with either six histidines at its C-ter (AAH6) or nine histidines at its N-ter (H9AA).
2. Materials 2.1. Chemicals
1. n-dodecyl-b-d-maltoside (DDM) was purchased from Anatrace. [14C]DDM was kindly provided by Marc Le Maire (CEA, CNRS, Université Paris-Sud 11, France). 2. Atractyloside (ATR) and carboxyatractyloside (CATR) were obtained from Sigma. 3. The nickel-nitrilotriacetic acid agarose matrix (Ni-NTA) for metal affinity chromatography is from Qiagen. It is provided as a 1:1 (vol/vol) suspension in 50% ethanol (vol/vol). 4. 3-Laurylamido-N,N¢-dimethylpropylaminoxide (LAPAO) was synthesized as described in (5). It can now be purchased from Anatrace. 5. Hydroxyapatite Bio-Gel® was purchased from Bio-Rad. 6. Thrombin was from Sigma and used as recommended by the supplier.
2.2. Buffer Compositions
1. Buffer A: 150 mM NaPi pH 7.3, 10% (w/v) glycerol, 0.1% (w/v) DDM.
Native Membrane Proteins vs. Yeast Recombinant
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2. Buffer B: 100 mM Na2SO
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