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

Nature Methods volume 3, pages 931–937 (2006)Cite this article

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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Figure 1: Identification of spin systems.
Figure 2: Resolution of ambiguous connectivity between clusters.
Figure 3: Distribution of δ-NOE that reflects the difference in the number of common NOEs shared by two adjacent amide protons and those by two nonadjacent amides.
Figure 4: Comparison of structures determined by NMR and X-ray methods.

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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.

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Author notes

  1. Yingqi Xu and Yu Zheng: These authors contributed equally to this work.

Authors and 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

Authors
  1. Yingqi Xu
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  2. Yu Zheng
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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.

Corresponding author

Correspondence to Daiwen Yang.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

2D TROSY-HSQC of non-deuterated MBP. (PDF 199 kb)

Supplementary Fig. 2

Distributions of peak signal-to-noise ratio for the 3D TROSY-HNCA experiments. (PDF 453 kb)

Supplementary Fig. 3

Detailed information on the backbone assignments. (PDF 188 kb)

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. (PDF 164 kb)

Supplementary Fig. 5

Pulse sequence for recording the 4D 13C,15N-edited NOESY. (PDF 91 kb)

Supplementary Table 1

NMR structural statistics for MBP and HbCO A. (PDF 121 kb)

Supplementary Table 2

Experimental parameters. (PDF 115 kb)

Supplementary Methods (PDF 111 kb)

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Xu, Y., Zheng, Y., Fan, JS. et al. A new strategy for structure determination of large proteins in solution without deuteration. Nat Methods 3, 931–937 (2006). https://doi.org/10.1038/nmeth938

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