Purification and biochemical characterization of the ATP synthase from Heliobacterium modesticaldum

Jay How Yang, Iosifina Sarrou, Jose M. Martin-Garcia, Shangji Zhang, Kevin Redding, Petra Fromme

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Heliobacterium modesticaldum is an anaerobic photosynthetic bacterium that grows optimally at pH 6-7 and 52°C and is the only phototrophic member of the Firmicutes phylum family (gram-positive bacteria with low GC content). The ATP synthase of H. modesticaldum was isolated and characterized at the biochemical and biophysical levels. The isolated holoenzyme exhibited the subunit patterns of F-type ATP synthases containing a 5-subunit hydrophilic F<inf>1</inf> subcomplex and a 3-subunit hydrophobic F<inf>0</inf> subcomplex. ATP hydrolysis by the isolated HF<inf>1</inf>F<inf>0</inf> ATP synthase was successfully detected after pretreatment with different detergents by an in-gel ATPase activity assay, which showed that the highest activity was detected in the presence of mild detergents such as LDAO; moreover, high catalytic activity in the gel was already detected after the initial incubation period of 0.5 h. In contrast, HF<inf>1</inf>F<inf>0</inf> showed extremely low ATPase activity in harsher detergents such as TODC. The isolated fully functional enzyme will form the basis for future structural studies.

Original languageEnglish (US)
Pages (from-to)1-8
Number of pages8
JournalProtein Expression and Purification
Volume114
DOIs
StatePublished - Jun 8 2015

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Adenosine Triphosphate
Detergents
Adenosine Triphosphatases
Gels
Holoenzymes
Anaerobic Bacteria
Base Composition
Gram-Positive Bacteria
Hydrolysis
Enzymes

Keywords

  • ATP hydrolysis
  • ATP synthase
  • Heliobacterium modesticaldum
  • In-gel function assay

ASJC Scopus subject areas

  • Biotechnology

Cite this

Purification and biochemical characterization of the ATP synthase from Heliobacterium modesticaldum. / Yang, Jay How; Sarrou, Iosifina; Martin-Garcia, Jose M.; Zhang, Shangji; Redding, Kevin; Fromme, Petra.

In: Protein Expression and Purification, Vol. 114, 08.06.2015, p. 1-8.

Research output: Contribution to journalArticle

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AU - Martin-Garcia, Jose M.

AU - Zhang, Shangji

AU - Redding, Kevin

AU - Fromme, Petra

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AB - Heliobacterium modesticaldum is an anaerobic photosynthetic bacterium that grows optimally at pH 6-7 and 52°C and is the only phototrophic member of the Firmicutes phylum family (gram-positive bacteria with low GC content). The ATP synthase of H. modesticaldum was isolated and characterized at the biochemical and biophysical levels. The isolated holoenzyme exhibited the subunit patterns of F-type ATP synthases containing a 5-subunit hydrophilic F1 subcomplex and a 3-subunit hydrophobic F0 subcomplex. ATP hydrolysis by the isolated HF1F0 ATP synthase was successfully detected after pretreatment with different detergents by an in-gel ATPase activity assay, which showed that the highest activity was detected in the presence of mild detergents such as LDAO; moreover, high catalytic activity in the gel was already detected after the initial incubation period of 0.5 h. In contrast, HF1F0 showed extremely low ATPase activity in harsher detergents such as TODC. The isolated fully functional enzyme will form the basis for future structural studies.

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