How to prepare membrane proteins for solid-state NMR: A case study on the alpha-helical integral membrane protein diacylglycerol kinase from E. coli

Chembiochem. 2005 Sep;6(9):1693-700. doi: 10.1002/cbic.200500054.

Abstract

Several studies have demonstrated that it is viable to use microcrystalline preparations of water-soluble proteins as samples in solid-state NMR experiments [1-5]. Here, we investigate whether this approach holds any potential for studying water-insoluble systems, namely membrane proteins. For this case study, we have prepared proteoliposomes and small crystals of the alpha-helical membrane-protein diacylglycerol kinase (DGK). Preparations were characterised by 13C- and 15N-cross-polarization magic-angle spinning (CPMAS) NMR. It was found that crystalline samples produce better-resolved spectra than proteoliposomes. This makes them more suitable for structural NMR experiments. However, reconstitution is the method of choice for biophysical studies by solid-state NMR. In addition, we discuss the identification of lipids bound to membrane-protein crystals by 31P-MAS NMR.

MeSH terms

  • Adenosine Triphosphate / pharmacology
  • Crystallization
  • Diacylglycerol Kinase / chemistry*
  • Diacylglycerol Kinase / metabolism
  • Escherichia coli / enzymology*
  • Lipids / chemistry
  • Membrane Proteins / chemistry*
  • Membrane Proteins / metabolism
  • Nuclear Magnetic Resonance, Biomolecular / methods*
  • Protein Structure, Secondary

Substances

  • Lipids
  • Membrane Proteins
  • Adenosine Triphosphate
  • Diacylglycerol Kinase