分析 ATP synthase 在粒線體磷脂雙層膜結構組成

分析 ATP synthase 在粒線體磷脂雙層膜結構組成 摘要 粒線體F1Fo-ATP 分子馬達能將能量氧化代謝合成ATP，無論如何，直接觀察粒腺體上酵素目前尚未有報告，這裡，我們用二種方法來對 ATP 分析，使用電子顯微鏡和AFM， 介紹 能量來自質子電化學氧化代謝反應，藉由mitochondrial ATP synthase 中質子驅動力合成ATP 材料方法: 2.1. Reconstitution of bovine F1Fo-ATPase into lipid bilayers Bovine F1Fo-ATPase was purified in 0.1% (w/v) n-dodecyl- b-D-maltoside (DDM; Anatrace, OH, USA) as previously described (Buchanan and Walker, 1996). The inhibitor protein IF1 was stripped from the protein complex after addition of 1:1000 volumes of NH3OH at pH 9.2 for 15 min before loading into a Superose 6 gel filtration (Amersham Bioscience). Protein was eluted in a buffer consisting of 20 mM Tris–HCl, pH 8.0, 50 mM trehalose, 10% (w/v) glycerol, 100 mM NaCl, 2 mM MgSO4, 1mM EDTA, 1 mM DTT, 0.1% DDM, 0.001% PMSF. Where appropriate 20 mM Tris–HCl, pH 8.0 was replaced by 20 mM Hepes–NaOH, pH 7.3. Inhibition with 0.16 mM DCCD (dicyclohexyl-carbodiimide) was performed as described by Gibbons et al. (2000). In our experiments, 16 mM ATP was added to the protein solution before addition of DCCD.Purified bovine F1Fo-ATP synthase was added to and incubated with different phospholipids (Avanti Polar Lipids, Alabaster, AL, USA): egg yolk phosphatidylcholine, egg yolk PC; di-oleoyl-phosphatidylcholine, DOPC; egg yolk PC/DOPC mixtures and di-myristoyl-phosphatidylcholine, DMPC at different lipid-to-protein ratios (LPRs): 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.2, 1.5 and 3 (w/w) in a buffer consisting of 50 mM Hepes–NaOH, pH 7.3, 300 mM NaCl, 0.6 mM ADP, 1 mM AMP-PNP, 5 mM MgSO4, 0.02% (w/v) sodium azide, 0.001% PMSF. Lipids were prepared by chloroform evaporation under a stream of argon gas, solubilization in 1.5% DDM and sonication for 20–30 min. Final conditions were: 0.8–1 mg/ml F1Fo- ATPase, 0.4–0.5 mg/ml phospholipids (for an LPR of 0.5 (w/w)) and 0.4% DDM. Protein was incubated with phospholipids at 4 C for 2–3 h before detergent removal. Detergent was removed by addition of Bio-Beads SM2 (20–50 mesh) (Bio-Rad, CA, USA) at ratios of 15 mg of beads/mg detergent. Large (>1 lm) PLs and paracrystalline arrays appeared after 4–5 days. 2.2. Purification of S. cerevisiae F1Fo-ATPase Mitochondrial F1Fo-ATPase from S. cerevisiae was genetically engineered to introduce a hexahistidine-tag at the amino terminus of the mature b subunit as described by Mueller et al. (2004). Yeast was grown at 28 C for 48 h in YPEG medium (1% yeast extract, 2% peptone, 3% glycerol, and 2% ethanol) containing 0.05% Antifoam 204 (Sigma Chemical, St. Louis, MO, USA) and harvested by centrifugation at 5,000g. Cells were washed with cold distilled water and suspended in two volumes of breaking buffer (0.1 M Tris–HCl, pH 8.0, 0.65 M sorbitol, 5 mM EDTA, 5 mM e-amino caproic acid, 5 mM p-aminobenzamidine, and 0.2% bovine serum albumin). Yeast cells were broken using glass beads (0.45 mm) and Bead Beater (Biospec Products, Bartlesville, OK, USA). The cell debris was removed by two sequential centrifugations at 5,000g for 10 min. The mitochondria were isolated by centrifugation at 30,000g and washed twice with wash buffer (20 mM Tris–HCl, pH 7.5, 0.65 M sorbitol, 1 mM EDTA, 5 mM e-amino caproic acid, 5 mM p-aminobenzamidine, and 0.2% bovine serum albumin). Mitochondria suspensions at a protein concentration of 30 mg/ml were stored at 80 C until further use. For purification of yeast F1Fo-ATP synthase, mitochondria were thawed and resuspended at a protein concentration of 8.5 mg/ml in solubilization buffer (50 mM sodium phosphate, pH 7.5, 0.25 M sucrose, 5 mM e-amino caproic acid, 5 mM p-aminobenzamidine, and 1 mM EDTA). DDM was added dropwise from a 10% stock solution in water to a final concentration of 1%. The solution was stirred for 15 min and then centrifuged (31,000g, 45 min). The supernatant was adjusted to 15 mM imidazol and applied to a Ni–NTA Superflow column (Qiagen, Valencia, CA, USA) equilibrated with buffer A (50 mM sodium phosphate, pH 7.5, 0.25 M sucrose, 15 mM imidazole, 0.3 M NaCl, 5 mM e-amino caproic acid, 5 mM p-aminobenzamidine, 1 mMPMSF). Protein was eluted in the same buffer with a gradient of imidazole (0–300 mM). Fractions containing F1Fo-ATPase were pooled and concentrated to a volume of less than 1 ml using a centrifugal concentrator at 4 C (100 kDa molecular weight cut-off, Vivascience, Hannover, Germany) and further purified by gel filtration at 4 C on a HiLoad 16/100 Superose 6 (Amersham, Piscataway, NJ) column equilibrated with 50 mM Tris–HCl, pH 8.0, 50 mM trehalose, 10% (w/v) glycerol, 100 mM NaCl, 2 mM MgSO4, 1 mM EDTA, 1 mM DTT, 1 mM PMSF and 0.1% DDM. The protein quality in the fractions containing the F1Fo-ATP synthase was evaluated by SDS– PAGE. Fractions containing non-proteolyzed F1Fo-ATP synthase were pooled and stored at 20 C until further use. 2.3. 2D Crystallization of yeast F1Fo-ATPase 2D Crystallization trials were performed in three steps. First, 0.5 ll of Ni–NTA–DOGS lipid (1,2-dioleoyl-sn-glycero- 3-{[N-(5-amino-1-carboxypentyl)-imino-diacetic-acid]- succinyl} (nickel salt)) (Avanti Polar Lipids, Alabaster, AL, USA) at 0.1 mg/ml in chloroform:methanol 9:1 (v/v) was spread on a 70 ll droplet of detergent-free buffer (50 mM Hepes, pH 7.3, 300 mM NaCl, 5 mM MgCl2) in custom-designed Teflon wells (4 mm in diameter and 4 mm in depth) with an injection hole to allow addition of proteins or Bio-Beads as described by Levy et al. (1999). The mixture was incubated overnight at room temperature to form a lipid monolayer at the air–liquid interface. In a second step, yeast F1Fo-ATPase at 0.8 mg/ml was incubated with nucleotides (1 mM AMP-PNP, 0.1 mM ADP) for 20 min on ice. Phospholipids [8 ll, 10 mg/ml DOPC:DOPA 9:1 (v/v)] were added to 86 ll of protein solution (0.8 mg/ml) to a final LPR of 1.2 (w/w) and a final protein concentration of 0.7 mg/ml. DDM [6 ll, 10% (w/v) stock solution] was added to reach a final concentration 0.6% DDM. This ternary mixture (lipid, detergent and protein) was incubated overnight at 4 C and 8.2 ll injected into the Teflon well below the preformed lipid monolayer. Finally, after 4-h incubation to allow binding of the ternary mixture to the functionalized interface, detergent was removed by adding 5 mg Bio-Beads in the wells and incubation for 5–6 h, followed by a second addition of 5 mg Bio-Beads and overnight incubation at 20 C. 2.4. Atomic force microscopy Bovine F1Fo-ATP synthase reconstituted in egg yolk PC or egg yolk PC/DOPC (3:1) mixtures at 0.8 mg/ml protein concentration was diluted 10-fold in 20 mM Tris–HCl, pH 8, 150 mM NaCl, 5 mM MgCl2 (AFM buffer) and deposited on freshly cleaved muscovite mica. After 30–60 min of adsorption, the sample was gently washed with AFM buffer to remove membranes that were not firmly attached to the mica. AFM was performed in contact mode with a commercial Nanoscope Multimode microscope (Digital Instruments, Veeco Metrology Group) equipped with an infrared laser head, fluid cell, and oxide-sharpened silicon nitride cantilevers (OMCL-TR400PSA, Olympus). Topographs were acquired at minimal loading forces (6100 pN). For nanodissection (see Fig. 3F) a selected area was scanned with a force of 500–750 pN to remove the F1 moiety. Trace and retrace signals were recorded simultaneously at a line frequency of 4.7 Hz. Besides flattening and adjustment of the colour scale, no image processing was applied to AFM images. The perspective view of the AFM topograph in Fig. 3E was prepared using the software package supplied by the AFM manufacturer. 結果討論: 結論: 心得: 真是有夠難翻，只能自已在氣自已英文不好，