The Effect of Detergent, Temperature, and Lipid on the Oligomeric State of MscL Constructs: Insights from Mass Spectrometry

Eamonn Reading, Troy A Walton, Idlir Liko, Michael T Marty, Arthur Laganowsky, Douglas C Rees, Carol V Robinson

Research output: Contribution to journalArticlepeer-review

66 Citations (Scopus)
86 Downloads (Pure)

Abstract

The mechanosensitive channel of large conductance (MscL) acts as an emergency release valve for osmotic shock of bacteria preventing cell lysis. The large pore size, essential for function, requires the formation of oligomers with tetramers, pentamers, or hexamers observed depending on the species and experimental approach. We applied non-denaturing (native) mass spectrometry to five different homologs of MscL to determine the oligomeric state under more than 50 different experimental conditions elucidating lipid binding and subunit stoichiometry. We found equilibrium between pentameric and tetrameric species, which can be altered by detergent, disrupted by binding specific lipids, and perturbed by increasing temperature (37°C). We also established the presence of lipopolysaccharide bound to MscL and other membrane proteins expressed in Escherichia coli, revealing a potential source of heterogeneity. More generally, we highlight the use of mass spectrometry in probing membrane proteins under a variety of detergent-lipid environments relevant to structural biology.

Original languageEnglish
Pages (from-to)593-603
Number of pages11
JournalChemistry and Biology
Volume22
Issue number5
Early online date21 May 2015
DOIs
Publication statusPublished - 21 May 2015

Keywords

  • Amino Acid Sequence
  • Bacterial Proteins/chemistry
  • Detergents/chemistry
  • Escherichia coli/metabolism
  • Escherichia coli Proteins/chemistry
  • Ion Channels/chemistry
  • Lipids/chemistry
  • Lipopolysaccharides/chemistry
  • Mass Spectrometry
  • Molecular Sequence Data
  • Protein Multimerization
  • Protein Structure, Quaternary
  • Recombinant Fusion Proteins/biosynthesis
  • Sequence Alignment
  • Temperature

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