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Proc Natl Acad Sci U S A. 2018 Feb 20;115(8):E1710-E1719. doi: 10.1073/pnas.1717560115. Epub 2018 Feb 5.

15N detection harnesses the slow relaxation property of nitrogen: Delivering enhanced resolution for intrinsically disordered proteins.

Author information

1
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115.
2
Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215.
3
Molecular Profiling Research Center for Drug Discovery, National Institute of Advanced Industrial Science and Technology, 135-0064 Tokyo, Japan.
4
Molecular and Cellular Biochemistry Department, Indiana University, Bloomington, IN 47405.
5
Magnetic Resonance Spectroscopy NMR Application, Bruker BioSpin GmbH, 76287 Rheinstetten, Germany.
6
Conway Institute of Biomolecular and Biomedical Sciences, University College Dublin, Dublin 4, Ireland.
7
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115; gerhard_wagner@hms.harvard.edu hari@hms.harvard.edu.

Abstract

Studies over the past decade have highlighted the functional significance of intrinsically disordered proteins (IDPs). Due to conformational heterogeneity and inherent dynamics, structural studies of IDPs have relied mostly on NMR spectroscopy, despite IDPs having characteristics that make them challenging to study using traditional 1H-detected biomolecular NMR techniques. Here, we develop a suite of 3D 15N-detected experiments that take advantage of the slower transverse relaxation property of 15N nuclei, the associated narrower linewidth, and the greater chemical shift dispersion compared with those of 1H and 13C resonances. The six 3D experiments described here start with aliphatic 1H magnetization to take advantage of its higher initial polarization, and are broadly applicable for backbone assignment of proteins that are disordered, dynamic, or have unfavorable amide proton exchange rates. Using these experiments, backbone resonance assignments were completed for the unstructured regulatory domain (residues 131-294) of the human transcription factor nuclear factor of activated T cells (NFATC2), which includes 28 proline residues located in functionally important serine-proline (SP) repeats. The complete assignment of the NFATC2 regulatory domain enabled us to study phosphorylation of NFAT by kinase PKA and phosphorylation-dependent binding of chaperone protein 14-3-3 to NFAT, providing mechanistic insight on how 14-3-3 regulates NFAT nuclear translocation.

KEYWORDS:

15N detection; IDP; NFAT; NMR resonance assignment; nuclear localization

PMID:
29432148
PMCID:
PMC5828609
DOI:
10.1073/pnas.1717560115
[Indexed for MEDLINE]
Free PMC Article

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