The stator complex of the A1A0-ATP synthase--structural characterization of the E and H subunits.
Archaeal ATP synthase (A-ATPase) is the functional homolog to the ATP synthase found in bacteria, mitochondria and chloroplasts, but the enzyme is structurally more related to the proton-pumping vacuolar ATPase found in the endomembrane system of eukaryotes. We have cloned, overexpressed and characterized the stator-forming subunits E and H of the A-ATPase from the thermoacidophilic Archaeon, Thermoplasma acidophilum. Size exclusion chromatography, CD, matrix-assisted laser desorption ionization time-of-flight mass spectrometry and NMR spectroscopic experiments indicate that both polypeptides have a tendency to form dimers and higher oligomers in solution. However, when expressed together or reconstituted, the two individual polypeptides interact with high affinity to form a stable heterodimer. Analyses by gel filtration chromatography and analytical ultracentrifugation show the heterodimer to have an elongated shape, and the preparation to be monodisperse. Thermal denaturation analyses by CD and differential scanning calorimetry revealed the more cooperative unfolding transitions of the heterodimer in comparison to those of the individual polypeptides. The data are consistent with the EH heterodimer forming the peripheral stalk(s) in the A-ATPase in a fashion analogous to that of the related vacuolar ATPase.
Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY 13210, USA.
Erik
E
Kish-Trier
Lee-Ann K
LK
Briere
Stanley D
SD
Dunn
Stephan
S
Wilkens
D002152:Calorimetry, Differential Scanning; D002352:Carrier Proteins / Q000378:metabolism; D020134:Catalytic Domain; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003001:Cloning, Molecular; D019281:Dimerization; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D019605:Euryarchaeota / Q000201:enzymology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D058619:Maltose-Binding Proteins; D008958:Models, Molecular; D008970:Molecular Weight; D019906:Nuclear Magnetic Resonance, Biomolecular; D016366:Open Reading Frames; D011485:Protein Binding; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism; D011789:Quantum Theory; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012399:Rotation; D020539:Sequence Analysis, Protein; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014461:Ultracentrifugation; D014867:Water / Q000737:chemistry
D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D014867:Water; D006180:Proton-Translocating ATPases