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GFT projection NMR based resonance assignment of membrane proteins: application to subunit c of E. coli F1F0 ATP synthase in LPPG micelles

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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.

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Abbreviations

GFT:

G-matrix Fourier transformation

LPPG:

1-Palmitoyl-2-hydroxy-sn-glycero-3-[phospho-RAC-(1-glycerol)]

RAC:

Racemic

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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.

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Author notes

  1. Hanudatta S. Atreya

    Present address: NMR Research Centre, Indian Institute of Science, Bangalore, 560012, India

Authors and Affiliations

  1. Department of Chemistry, State University of New York at Buffalo, Buffalo, NY, 14260, USA

    Qi Zhang, Hanudatta S. Atreya & Thomas Szyperski

  2. Biochemistry Department, Albert Einstein College of Medicine, New York, NY, 10461, USA

    Douglas E. Kamen & Mark E. Girvin

Authors
  1. Qi Zhang
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  2. Hanudatta S. Atreya
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  3. Douglas E. Kamen
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  4. Mark E. Girvin
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  5. Thomas Szyperski
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Corresponding author

Correspondence to Thomas Szyperski.

Additional information

Qi Zhang, Hanudatta S. Atreya, Douglas E. Kamen, Mark E. Girvin and Thomas Szyperski—New York Consortium on Membrane Protein Structure.

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Zhang, Q., Atreya, H.S., Kamen, D.E. et al. GFT projection NMR based resonance assignment of membrane proteins: application to subunit c of E. coli F1F0 ATP synthase in LPPG micelles. J Biomol NMR 40, 157–163 (2008). https://doi.org/10.1007/s10858-008-9224-8

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  • DOI: https://doi.org/10.1007/s10858-008-9224-8

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