title: How to prepare membrane proteins for solid-state NMR: A case study on the alpha-helical integral membrane protein diacylglycerol kinase from E. coli.
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.
authors.affiliation: Centre for Biomolecular Magnetic Resonance and Institut für Biophysikalische Chemie, J. W. Goethe Universität, Marie-Curie-Strasse 9, 60439 Frankfurt, Germany.
authors.forename: Mark
authors.initials: M
authors.lastname: Lorch
authors.forename: Salem
authors.initials: S
authors.lastname: Faham
authors.forename: Christoph
authors.initials: C
authors.lastname: Kaiser
authors.forename: Ingrid
authors.initials: I
authors.lastname: Weber
authors.forename: A James
authors.initials: AJ
authors.lastname: Mason
authors.forename: James U
authors.initials: JU
authors.lastname: Bowie
authors.forename: Clemens
authors.initials: C
authors.lastname: Glaubitz
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D003460:Crystallization; D019852:Diacylglycerol Kinase / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D008055:Lipids / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D017433:Protein Structure, Secondary
chemical_list: D008055:Lipids; D008565:Membrane Proteins; D000255:Adenosine Triphosphate; D019852:Diacylglycerol Kinase