Design, purification and characterization of a soluble variant of the integral membrane protein MotB for structural studies

Daniel A. Andrews, Meng Xie, Victoria Hughes, Matthew C. Wilce, Anna Roujeinikova

Research output: Contribution to journalArticleResearchpeer-review

4 Citations (Scopus)

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.
Original languageEnglish
Number of pages9
JournalJournal of the Royal Society Interface
Volume10
Issue number79
DOIs
Publication statusPublished - 6 Feb 2013

Keywords

  • flagellar motor
  • membrane proteins
  • motility protein B
  • protein engineering

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