期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2018
卷号：115
期号：8
页码：E1710-E1719
DOI：10.1073/pnas.1717560115
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：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.
Loading...
