GFT projection NMR based resonance assignment of membrane proteins: application to subunit c of E. coli F sub(1)F sub(0) ATP synthase in LPPG micelles

• Published in: Journal of biomolecular NMR Vol. 40; no. 3; pp. 157 - 163
• Main Authors: Zhang, Qi, Atreya, Hanudatta S, Kamen, Douglas E, Girvin, Mark E, Szyperski, Thomas
• Format: Journal Article
• Language: English
• Published: 01-03-2008
• Subjects: Escherichia coli
• ISSN: 0925-2738
• DOI: 10.1007/s10858-008-9224-8

G-matrix FT projection NMR spectroscopy was employed for resonance assignment of the 79-residue subunit c of the Escherichia coli F sub(1)F sub(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 super( alpha beta )C super( alpha ), L-(4,3)D HNN(CO)C super( alpha beta )C super( alpha ), (4,2)D HACA(CO)NHN and (4,3)D HCCH, were acquired along with simultaneous 3D super(15)N, super(13)C super(aliphatic), super(13)C super(aromatic)-resolved [ super(1)H, super(1)H]-NOESY with a total measurement time of similar to 43 h. Data analysis resulted in sequence specific assignments for all routinely measured backbone and super(13)C super( beta ) shifts, and for 97% of the side chain shifts. Moreover, the use of two G super(2)FT NMR experiments, that is, (5,3)D HN{N,CO}{C super( alpha beta )C super( alpha )} and (5,3)D {C super( alpha beta )C super( 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 super(2)FT NMR experiments enables one to efficiently obtain (nearly) complete resonance assignments of membrane proteins.