doi:10.1016/j.bbamem.2007.12.017
Copyright © 2008 Elsevier B.V. All rights reserved.
Remarkable stability of the proton translocating F1FO-ATP synthase from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1
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Tina Suhaia, Norbert A. Denchera, Ansgar Poetschb and Holger Seelerta, 
, 
aPhysical Biochemistry, Department of Chemistry, Darmstadt University of Technology, Petersenstrasse 22, D-64287 Darmstadt, Germany
bBiochemistry of Plants, Ruhr-University Bochum, Universitätsstrasse 150, D-44801 Bochum, Germany
Received 4 October 2007;
revised 28 November 2007;
accepted 13 December 2007.
Available online 31 December 2007.
Abstract
For functional characterization, we isolated the F1FO-ATP synthase of the thermophilic cyanobacterium Thermosynechococcus elongatus. Because of the high content of phycobilisomes, a combination of dye-ligand chromatography and anion exchange chromatography was necessary to yield highly pure ATP synthase. All nine single F1FO subunits were identified by mass spectrometry. Western blotting revealed the SDS stable oligomer of subunits c in T. elongatus. In contrast to the mass archived in the database (10,141 Da), MALDI-TOF-MS revealed a mass of the subunit c monomer of only 8238 Da. A notable feature of the ATP synthase was its ability to synthesize ATP in a wide temperature range and its stability against chaotropic reagents. After reconstitution of F1FO into liposomes, ATP synthesis energized by an applied electrochemical proton gradient demonstrated functional integrity. The highest ATP synthesis rate was determined at the natural growth temperature of 55 °C, but even at 95 °C ATP production occurred. In contrast to other prokaryotic and eukaryotic ATP synthases which can be disassembled with Coomassie dye into the membrane integral and the hydrophilic part, the F1FO-ATP synthase possessed a particular stability. Also with the chaotropic reagents sodium bromide and guanidine thiocyanate, significantly harsher conditions were required for disassembly of the thermophilic ATP synthase.
Keywords: ATP synthase; Cyanobacteria; Thermo stability; Chaotropic reagent
Abbreviations: BN-PAGE, Blue-native polyacrylamide gel electrophoresis; CF1FO, chloroplast ATP synthase; chl a, chlorophyll a; DCCD, dicyclohexylcarbodiimide; DDM, n-dodecyl-β-d-maltoside; ESI-MS, electrospray ionisation mass spectrometry; MALDI-TOF-MS, matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry
Fig. 1. BN-PAGE (3.5–16% gradient of polyacrylamide) of the purification steps of F1FO from thylakoid membranes of Thermosynechococcus elongatus. (A) unstained gel; (B) same gel but additionally Coomassie R-250-stained. Lane 1, suspended pellet after 50% ammonium sulfate precipitation; lane 2, typical fraction of the density gradient containing 38% sucrose; lane 3, F1FO after Reactive Red 120 – chromatography; lane 4, F1FO after anion exchange – chromatography; lane 5 shows the complete CF1FO of Spinacea oleracea and its two parts F1 and FO. As standard a high molecular mass marker (in kDa) is indicated on the left side. Purification is demonstrated by decreasing protein contamination in comparison to the F1FO content.
Fig. 2. (A) Silver-stained SDS-PAGE (14%) of purified CF1FO of Spinacea oleracea (lane 1) and F1FO of Thermosynechococcus elongatus (lane 2). All subunits of F1FO except subunits c and a were identified with MALDI-TOF-MS PMF. Subunit a was identified with ESI-MS/MS. (B) Western blot of a SDS-PAGE with polyclonal antibodies raised against monomer III from Sp. oleracea. Lane 1, oligomer and monomer of the subunit III of CF1FO of Sp. oleracea; lane 2, oligomer and monomer of subunit c of T. elongatus. (C) MALDI-MS analysis of intact c monomer. The identified mass is indicated. Subunit III of CF1FO of spinach was used as external standard.
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Fig. 3. (A) ATP synthesis activity of F1FO of Thermosynechococcus elongatus measured with the “one-step method”. The graph reflects the change in ATP produced, initiated by the established ΔpH/ΔΨ. The steep increase of the curve after 190 s is caused by adding the ATP standard. The curve of the DCCD inhibited sample is shown as dotted line, demonstrating that no ATP is produced. (B) ATP synthesis rate of F1FO of T. elongatus at different temperatures measured with the “two-step method”. For calculation of the ATP synthesis rate, the total amount of ATP produced within 10 s was quantified. Highest ATP synthesis rate could be observed at 55 °C. The average ATP synthesis rate of the DCCD inhibited ATP synthases incubated at 75 °C, 85 °C and 95 °C is shown (ØDCCD). (C) ATP synthesis rate of F1FO of T. elongatus at 55 °C over a time period of 6 h measured with the “two-step method”. The ATP synthesis rate remains constant at a high level for 4 h of incubation. Longer incubation leads to a decrease of the ATP synthesis activity.
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Fig. 4. Stability of F1FO of Thermosynechococcus elongatus. (A) Western blot of a BN-gel showing the stability of the F1FO-ATP synthase of Thermosynechococcus elongatus towards Coomassie dye. Lane 1 CF1FO of Spinacea oleracea, lanes 2–9 F1FO with different concentrations of Coomassie Serva blue G (2) 0% (w/v), (3) 1.0% (w/v), (4) 1.5% (w/v), (5) 2.0% (w/v), (6) 2.5% (w/v), (7) 3.0% (w/v), (8) 3.5% (w/v), (9) 4.0% (w/v). The stability of the F1FO-ATP synthase was not strongly affected by the Coomassie dye. In contrast to the reference CF1FO, F1FO is stable up to 3.5% Coomassie dye. Only the highest dye concentration of 4.0% induced the disaggregation of the F1FO-ATP synthase in its F1 and FO part. (B) Stability of F1FO of Thermosynechococcus elongatus towards sodium bromide and guanidine thiocyanate at various temperatures. Western blot of spinach CF1FO and F1FO incubated in 250 mM guanidine thiocyanate at the different temperatures. The Western blots demonstrate the higher stability of the F1FO-ATP synthase at higher temperatures compared to CF1FO. R, reference ATP synthases. (C) Effect of chaotropic reagents on the stability of the F1FO-ATP synthase at different temperatures. The percentage of F1 compared to the total amount of ATP synthase is shown. The fraction of the F1 part increases at 56 °C, indicating the starting disassembly of F1FO.
Table 1.
Purification of the F1FO-ATP synthase of Thermosynechococcus elongatus
Table 2.
Subunits of the ATP synthase of Thermosynechococcus elongatus identified with MALDI-TOF-MS PMF after tryptic digestion
a NCBInr database accession number.
b Scores obtained by “Mascot”. Scores of 70 and above are considered as significant.
c Theoretic values from mature proteins, i.e., without transit peptides.
Table 3.
Identification of the ATP synthase subunit a of Thermosynechococcus elongatus by nLC-ESI-MS/MS after tryptic digestion
a The amino acid residues appearing before and after the dot correspond to residues preceding and following the peptide in the protein sequence.
b Monoisotopic mass.
c Cross-correlation score is based on comparison of the MS/MS data to the theoretical distribution of ions produced for the peptide.
d Calculated difference between the top Xcorr values for the given peptide.
e Total number of b and y ions identified/theoretical.
Corresponding author. Tel.: +49 6151 165193; fax: +49 6151 164171.
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