title: Design, purification and characterization of a soluble variant of the integral membrane protein MotB for structural studies.
abstract: The bacterial flagellar motor is an intricate nanomachine powered by a transmembrane electrochemical gradient. Rotation is driven by the cumulative action of several peptidoglycan-anchored stator complexes on the rotor. In proton-motive force-driven motors, the stator complex is composed of a motility protein B (MotB) dimer surrounded by four copies of MotA, where both MotA and MotB are integral membrane proteins. The lack of full-length MotA and MotB structures hinders understanding of the mechanism of torque generation. Given the low levels of expression and low stability of detergent-solubilized MotB, a soluble chimaeric variant was engineered, where the two transmembrane helices of the MotB dimer were replaced by a leucine zipper. The biochemical and biophysical analysis of the resultant protein showed that it was properly folded, stable, behaved as a monodisperse dimer at low pH, had molecular dimensions close to those expected for native MotB and yielded reproducible crystals. The chimaeric protein is, therefore, a good candidate for structural studies. This 'solubilization by design' approach may be generally applicable to the production of soluble forms of other dimeric, trimeric and tetrameric single-span membrane proteins for functional and structural studies.
authors.affiliation: Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Andrews
authors.forename: Meng
authors.initials: M
authors.lastname: Xie
authors.forename: Victoria
authors.initials: V
authors.lastname: Hughes
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Wilce
authors.forename: Anna
authors.initials: A
authors.lastname: Roujeinikova
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003460:Crystallization; D017931:DNA Primers / Q000235:genetics; D008958:Models, Molecular; D020409:Molecular Motor Proteins / Q000737:chemistry* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D020409:Molecular Motor Proteins; C064779:MotB protein, Bacteria; D011994:Recombinant Proteins