GFT projection NMR based resonance assignment of membrane proteins: application to subunit C of E. coli F(1)F (0) ATP synthase in LPPG micelles.
G-matrix FT projection NMR spectroscopy was employed for resonance assignment of the 79-residue subunit c of the Escherichia coli F(1)F(0) 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 (alphabeta) C (alpha), L-(4,3)D HNN(CO)C (alphabeta) C (alpha), (4,2)D HACA(CO)NHN and (4,3)D HCCH, were acquired along with simultaneous 3D (15)N, (13)C(aliphatic), (13)C(aromatic)-resolved [(1)H,(1)H]-NOESY with a total measurement time of approximately 43 h. Data analysis resulted in sequence specific assignments for all routinely measured backbone and (13)C(beta) shifts, and for 97% of the side chain shifts. Moreover, the use of two G(2)FT NMR experiments, that is, (5,3)D HN{N,CO}{C (alphabeta) C (alpha)} and (5,3)D {C (alphabeta) C (alpha)}{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 G(2)FT NMR experiments enables one to efficiently obtain (nearly) complete resonance assignments of membrane proteins.
Zhang
Qi
Q
Department of Chemistry, State University of New York at Buffalo, Buffalo, NY 14260, USA.
Atreya
Hanudatta S
HS
Kamen
Douglas E
DE
Girvin
Mark E
ME
Szyperski
Thomas
T
D025243:Bacterial Proton-Translocating ATPases / Q000737:chemistry*; D001330:Electronic Data Processing; D004926:Escherichia coli / Q000201:enzymology; D006017:Glycolipids / Q000494:pharmacology*; D007295:Inositol Phosphates / Q000494:pharmacology*; D008565:Membrane Proteins / Q000737:chemistry*; D008823:Micelles; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry
D006017:Glycolipids; D007295:Inositol Phosphates; C111513:LPPG glycoinositolphosphoceramide; D008565:Membrane Proteins; D008823:Micelles; D021122:Protein Subunits; D025243:Bacterial Proton-Translocating ATPases