An optimized purification and reconstitution method for the MscS channel: strategies for spectroscopical analysis

Biochemistry. 2007 Jun 12;46(23):6766-73. doi: 10.1021/bi700322k. Epub 2007 May 15.


The mechanosensitive channel of small conductance (MscS) plays a critical role in the osmoregulation of prokaryotic cells. The crystal structure of MscS revealed a homoheptamer with three transmembrane segments and a large cytoplasmic domain. It has been suggested that the crystal structure depicts an open state, but its actual functional conformation remains controversial. In the pursuit of spectroscopical approaches to MscS gating, we determined that standard purification methods yield two forms of MscS, with a considerable amount of unfolded channel. Here, we present an improved high-yield purification method based on Escherichia coli expression and a biochemical characterization of the reconstituted channel, optimized to yield approximately 4 mg of a single monodisperse product. Upon reconstitution into lipid vesicles, MscS is unusually prone to lateral aggregation depending on the lipid composition, particularly after sample freezing. Strategies for minimizing MscS aggregation in two dimensions for spectroscopic analyses of gating have been developed.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Crystallography, X-Ray
  • Electron Spin Resonance Spectroscopy
  • Escherichia coli / physiology
  • Escherichia coli Proteins / chemistry*
  • Escherichia coli Proteins / isolation & purification
  • Escherichia coli Proteins / metabolism
  • Ion Channels / chemistry*
  • Ion Channels / isolation & purification
  • Ion Channels / metabolism
  • Kinetics
  • Liposomes
  • Models, Molecular
  • Protein Conformation
  • Software


  • Escherichia coli Proteins
  • Ion Channels
  • Liposomes
  • MscS protein, E coli