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Improving the Accuracy of NMR Structures of Large Proteins Using Pseudocontact Shifts as Long-Range Restraints

Journal of Biomolecular NMR volume 28, Article number: 205 (2004) Cite this article

Abstract

We demonstrate improved accuracy in protein structure determination for large (≥30 kDa), deuterated proteins (e.g. STAT4NT) via the combination of pseudocontact shifts for amide and methyl protons with the available NOEs in methyl-protonated proteins. The improved accuracy is cross validated by Q-factors determined from residual dipolar couplings measured as a result of magnetic susceptibility alignment. The paramagnet is introduced via binding to thiol-reactive EDTA, and multiple sites can be serially engineered to obtain data from alternative orientations of the paramagnetic anisotropic susceptibility tensor. The technique is advantageous for systems where the target protein has strong interactions with known alignment media.

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Author notes

  1. Siddhartha P. Sarma

    Present address: Molecular Biophysics Unit, Indian Institute of Science, Bangalore Karnataka -, 560012, India

Affiliations

  1. Structural Biophysics Laboratory, National Cancer Institute, P.O. Box B, Frederick, MD, 21702-1201, U.S.A.

    Vadim Gaponenko, Amanda S. Altieri, Jess Li & R. Andrew Byrd

  2. Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, NC, 27157-1016, U.S.A.

    David A. Horita

Authors
  1. Vadim Gaponenko
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  2. Siddhartha P. Sarma
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  3. Amanda S. Altieri
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  4. David A. Horita
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  5. Jess Li
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  6. R. Andrew Byrd
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Gaponenko, V., Sarma, S.P., Altieri, A.S. et al. Improving the Accuracy of NMR Structures of Large Proteins Using Pseudocontact Shifts as Long-Range Restraints. J Biomol NMR 28, 205 (2004). https://doi.org/10.1023/B:JNMR.0000013706.09264.36

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  • DOI: https://doi.org/10.1023/B:JNMR.0000013706.09264.36

  • deuteration
  • ILV-labeling
  • NMR structural accuracy
  • pseudocontact shifts
  • residual dipolar coupling
  • STAT4