title: Purification and biochemical characterization of the ATP synthase from Heliobacterium modesticaldum.
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 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 0.5h. 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.
authors.affiliation: Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287-1604, USA; Center for Applied Structural Discovery, The Biodesign Institute, Arizona State University, Tempe, AZ 85287-1604, USA.
authors.forename: Jay-How
authors.initials: JH
authors.lastname: Yang
authors.affiliation: Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287-1604, USA; Institute of Molecular Biology & Biotechnology, Foundation for Research & Technology-Hellas, Nikolaou Plastira 100, GR-70013 Heraklion, Crete, Greece.
authors.forename: Iosifina
authors.initials: I
authors.lastname: Sarrou
authors.affiliation: Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287-1604, USA; Center for Applied Structural Discovery, The Biodesign Institute, Arizona State University, Tempe, AZ 85287-1604, USA.
authors.forename: Jose M
authors.initials: JM
authors.lastname: Martin-Garcia
authors.affiliation: School of Life Sciences, Arizona State University, Tempe, AZ 85287-1604, USA.
authors.forename: Shangji
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287-1604, USA.
authors.forename: Kevin E
authors.initials: KE
authors.lastname: Redding
authors.affiliation: Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287-1604, USA; Center for Applied Structural Discovery, The Biodesign Institute, Arizona State University, Tempe, AZ 85287-1604, USA. Electronic address: pfromme@asu.edu.
authors.forename: Petra
authors.initials: P
authors.lastname: Fromme
mesh_terms: D000255:Adenosine Triphosphate / Q000032:analysis / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D000068016:Eubacteriales / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases
keywords: ATP hydrolysis; ATP synthase; Heliobacterium modesticaldum; In-gel function assay