Bacterial production and solution NMR studies of a viral membrane ion channel

Methods Mol Biol. 2012;831:165-79. doi: 10.1007/978-1-61779-480-3_10.

Abstract

Advances in solution nuclear magnetic resonance (NMR) methodology that enable studies of very large proteins have also paved the way for studies of membrane proteins that behave like large proteins due to the added weight of surfactants. Solution NMR has been used to determine the high-resolution structures of several small, membrane proteins dissolved in detergent micelles and small bicelles. However, the usual difficulties with membrane proteins in producing, purifying, and stabilizing the proteins away from native membranes remain, requiring intensive screening efforts. Low levels of heterologous expression can be the most detrimental aspect to studying membrane proteins. This is exacerbated for NMR studies because of the costs of isotopically enriched media. Thus, solution NMR studies have tended to focus on relatively small, membrane proteins that can be expressed into inclusion bodies and refolded. Here, we describe the methods used to produce, purify, and refold the proton channel M2 into detergent micelles, and the procedures used to determine chemical shift assignments and the atomic level structure of the closed form of the homotetrameric channel.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Bioreactors*
  • Carbon Isotopes / metabolism
  • Escherichia coli / metabolism*
  • Models, Molecular*
  • Nitrogen Isotopes / metabolism
  • Nuclear Magnetic Resonance, Biomolecular / methods*
  • Protein Conformation
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / chemistry*
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / metabolism
  • Tryptophan / chemistry
  • Tryptophan / metabolism
  • Viral Matrix Proteins / biosynthesis
  • Viral Matrix Proteins / chemistry*
  • Viral Matrix Proteins / isolation & purification
  • Viral Matrix Proteins / metabolism

Substances

  • Carbon Isotopes
  • M2 protein, Influenza A virus
  • Nitrogen Isotopes
  • Recombinant Fusion Proteins
  • Viral Matrix Proteins
  • Tryptophan