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Full-Text Articles in Biochemistry
Tethering Polypeptides Through Bifunctional Peg Cross-Linking Agents To Probe Protein Function: Application To Atp Synthase., Daniel J Cipriano, Stanley D Dunn
Tethering Polypeptides Through Bifunctional Peg Cross-Linking Agents To Probe Protein Function: Application To Atp Synthase., Daniel J Cipriano, Stanley D Dunn
Biochemistry Publications
Chemical crosslinking mediated by short bifunctional reagents has been widely used for determining physical relationships among polypeptides in multisubunit proteins, but less often for functional studies. Here we introduce the approach of tethering polypeptides by using bifunctional reagents containing a lengthy, flexible PEG linker as a form of crosslinking especially suited to functional analyses. The rotary molecular motor ATP synthase was used as a model subject. Single cysteine residues were introduced into selected positions of ATP synthase epsilon subunit, a component of the rotor subcomplex of the enzyme, and the unrelated maltose binding protein (MBP), then the two purified recombinant …
The Stator Complex Of The A1a0-Atp Synthase--Structural Characterization Of The E And H Subunits., Erik Kish-Trier, Lee-Ann K Briere, Stanley D Dunn, Stephan Wilkens
The Stator Complex Of The A1a0-Atp Synthase--Structural Characterization Of The E And H Subunits., Erik Kish-Trier, Lee-Ann K Briere, Stanley D Dunn, Stephan Wilkens
Biochemistry Publications
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 …