TY - JOUR
T1 - GFT projection NMR based resonance assignment of membrane proteins
T2 - Application to subunit c of E. coli F1F0 ATP synthase in LPPG micelles
AU - Zhang, Qi
AU - Atreya, Hanudatta S.
AU - Kamen, Douglas E.
AU - Girvin, Mark E.
AU - Szyperski, Thomas
N1 - Funding Information:
Acknowledgements This work was supported by the National Institutes of Health (U54 GM074958-01) and the National Science Foundation (MCB 0416899). We thank Mr. Yu-Chieh Lin for help with the NMR sample preparation.
PY - 2008/3
Y1 - 2008/3
N2 - 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 HN NCO, L-(4,3)D HNN Cαβ Cα, L-(4,3)D HNN(CO)Cαβ Cα, (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{NCO} {CαβCα} and (5,3)D Cαβ Cα} {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.
AB - 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 HN NCO, L-(4,3)D HNN Cαβ Cα, L-(4,3)D HNN(CO)Cαβ Cα, (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{NCO} {CαβCα} and (5,3)D Cαβ Cα} {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.
KW - FFATP synthase
KW - GFT NMR
KW - LPPG
KW - Membrane protein
KW - Projection NMR
KW - Resonance assignment
KW - Subunitc
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U2 - 10.1007/s10858-008-9224-8
DO - 10.1007/s10858-008-9224-8
M3 - Article
C2 - 18273680
AN - SCOPUS:41149103328
SN - 0925-2738
VL - 40
SP - 157
EP - 163
JO - Journal of Biomolecular NMR
JF - Journal of Biomolecular NMR
IS - 3
ER -