1. GFT projection NMR based resonance assignment of membrane proteins: application to subunit c of E. coli F1F0 ATP synthase in LPPG micelles
- Author
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Douglas E. Kamen, Thomas Szyperski, Q. Zhang, Mark E. Girvin, and Hanudatta S. Atreya
- Subjects
Protein Conformation ,Stereochemistry ,Inositol Phosphates ,Protein subunit ,Analytical chemistry ,medicine.disease_cause ,Biochemistry ,Micelle ,Escherichia coli ,medicine ,Side chain ,Nuclear Magnetic Resonance, Biomolecular ,Micelles ,Spectroscopy ,Electronic Data Processing ,ATP synthase ,biology ,Chemistry ,Membrane Proteins ,Resonance ,Nuclear magnetic resonance spectroscopy ,Protein Subunits ,Membrane protein ,Bacterial Proton-Translocating ATPases ,biology.protein ,Glycolipids - Abstract
G-matrix FT projection NMR spectroscopy was employed for resonance assignment of the 79-residue subunit c of the Escherichia coli F1F0 ATP synthase embedded in micelles formed by lyso palmitoyl phosphatidyl glycerol (LPPG). Five GFT NMR experiments, that is, (3,2)D HNNCO, L-(4,3)D HNNC αβ C α, L-(4,3)D HNN(CO)C αβ C α, (4,2)D HACA(CO)NHN and (4,3)D HCCH, were acquired along with simultaneous 3D 15N, 13Caliphatic, 13Caromatic-resolved [1H,1H]-NOESY with a total measurement time of ∼43 h. Data analysis resulted in sequence specific assignments for all routinely measured backbone and 13Cβ shifts, and for 97% of the side chain shifts. Moreover, the use of two G2FT NMR experiments, that is, (5,3)D HN{N,CO}{C αβ C α} and (5,3)D {C αβ C α}{CON}HN, was explored to break the very high chemical shift degeneracy typically encountered for membrane proteins. It is shown that the 4D and 5D spectral information obtained rapidly from GFT and G2FT NMR experiments enables one to efficiently obtain (nearly) complete resonance assignments of membrane proteins.
- Published
- 2008