title: Magic-angle spinning solid-state NMR of a 144 kDa membrane protein complex: E. coli cytochrome bo3 oxidase.
abstract: Recent progress in magic-angle spinning (MAS) solid-state NMR (SSNMR) has enabled multidimensional studies of large, macroscopically unoriented membrane proteins with associated lipids, without the requirement of solubility that limits other structural techniques. Here we present initial sample preparation and SSNMR studies of a 144 kDa integral membrane protein, E. coli cytochrome bo(3) oxidase. The optimized protocol for expression and purification yields approximately 5 mg of the enzymatically active, uniformly (13)C,(15)N-enriched membrane protein complex from each liter of growth medium. The preparation retains endogenous lipids and yields spectra of high sensitivity and resolution, consistent with a folded, homogenous protein. Line widths of isolated signals are less than 0.5 ppm, with a large number of individual resonances resolved in the 2D and 3D spectra. The (13)C chemical shifts, assigned by amino acid type, are consistent with the secondary structure previously observed by diffraction methods. Although the structure is predominantly helical, the percentage of non-helical signals varies among residue types; these percentages agree well between the NMR and diffraction data. Samples show minimal evidence of degradation after several weeks of NMR data acquisition. Use of a triple resonance scroll resonator probe further improves sample stability and enables higher power decoupling, higher duty cycles and more advanced 3D experiments to be performed. These initial results in cytochrome bo(3) oxidase demonstrate that multidimensional MAS SSNMR techniques have sufficient sensitivity and resolution to interrogate selected parts of a very large uniformly (13)C,(15)N-labeled membrane protein.
authors.affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: Heather L
authors.initials: HL
authors.lastname: Frericks
authors.forename: Donghua H
authors.initials: DH
authors.lastname: Zhou
authors.forename: Lai Lai
authors.initials: LL
authors.lastname: Yap
authors.forename: Robert B
authors.initials: RB
authors.lastname: Gennis
authors.forename: Chad M
authors.initials: CM
authors.lastname: Rienstra
mesh_terms: D000595:Amino Acid Sequence; D002247:Carbon Isotopes; D003576:Electron Transport Complex IV / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008958:Models, Molecular; D008970:Molecular Weight; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012680:Sensitivity and Specificity; D013696:Temperature; D013997:Time Factors; D014961:X-Ray Diffraction
chemical_list: D002247:Carbon Isotopes; D008565:Membrane Proteins; D009587:Nitrogen Isotopes; D003576:Electron Transport Complex IV