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A new strategy for structure determination of large proteins in solution without deuteration

Nature Methods volume 3, pages 931937 (2006) | Download Citation

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Abstract

So far high-resolution structure determination by nuclear magnetic resonance (NMR) spectroscopy has been limited to proteins <30 kDa, although global fold determination is possible for substantially larger proteins. Here we present a strategy for assigning backbone and side-chain resonances of large proteins without deuteration, with which one can obtain high-resolution structures from 1H-1H distance restraints. The strategy uses information from through-bond correlation experiments to filter intraresidue and sequential correlations from through-space correlation experiments, and then matches the filtered correlations to obtain sequential assignment. We demonstrate this strategy on three proteins ranging from 24 to 65 kDa for resonance assignment and on maltose binding protein (42 kDa) and hemoglobin (65 kDa) for high-resolution structure determination. The strategy extends the size limit for structure determination by NMR spectroscopy to 42 kDa for monomeric proteins and to 65 kDa for differentially labeled multimeric proteins without the need for deuteration or selective labeling.

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Acknowledgements

This research was supported by a grant from the Biomedical Research Council (BMRC), and Agency for Science, Technology and Research, A*Star of Singapore. D.Y. is the recipient of a BMRC Young Investigator Award. The authors thank L.E. Kay (University of Toronto) and C. Ho (Carnegie Mellon University) for providing the MBP and HbCO A samples, respectively.

Author information

Author notes

    • Yingqi Xu
    •  & Yu Zheng

    These authors contributed equally to this work.

Affiliations

  1. Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543.

    • Yingqi Xu
    • , Yu Zheng
    • , Jing-Song Fan
    •  & Daiwen Yang

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Contributions

D.Y. designed the research study; Y.X., Y.Z., J.-S.F. and D.Y. performed the research; Y.X. analyzed the MBP data; Y.Z. assigned DdCAD-1 and HbCO A; J.-S.F. determined the structure of HbCO A; Y.X. and D.Y. wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Daiwen Yang.

Supplementary information

PDF files

  1. 1.

    Supplementary Fig. 1

    2D TROSY-HSQC of non-deuterated MBP.

  2. 2.

    Supplementary Fig. 2

    Distributions of peak signal-to-noise ratio for the 3D TROSY-HNCA experiments.

  3. 3.

    Supplementary Fig. 3

    Detailed information on the backbone assignments.

  4. 4.

    Supplementary Fig. 4

    Relative peak intensity as a function of overall correlation time for different types of correlations in a number of 3D and 4D spectra.

  5. 5.

    Supplementary Fig. 5

    Pulse sequence for recording the 4D 13C,15N-edited NOESY.

  6. 6.

    Supplementary Table 1

    NMR structural statistics for MBP and HbCO A.

  7. 7.

    Supplementary Table 2

    Experimental parameters.

  8. 8.

    Supplementary Methods

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DOI

https://doi.org/10.1038/nmeth938

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